Issued  March  12, 1907. 


^        U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  No.  95. 
A.  D.  MELVIN,  Chief  op  Buhbau.  . 


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THE  BACTERIOLYTIC  POWER  OF  THE 
BLOOD  SERUM  OF  HOGS. 


BY 

B.  M.  BOLTON,  M.  D., 

Bacteriologist,  Biochemic  Division,  Bureau  of  Animal  Industry. 


'^^^^'> 

<>/■  ^'^Z 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 

1907. 


Issued  March  12, 1907. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  No.  95. 
A.  D.  MELVIN,  Chief  of  Bureau. 


THE  BACTERIOLYTIC  POWER  OF  THE 
BLOOD  SERUM  OF  HOGS. 


BY 
B.  M.  BOLTON.   M.  D., 

Bacteriologist,  Biocheniic  Division,  Bureau  of  Animal  Industry. 


WASHINGTON: 

GOVERNMENT    PRINTING   OFFICE. 

1907. 


BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  P.  Mkuvin. 

An*ijUiiht  Chirf:   A.  M.  Karrin«}TI)N. 

Oiirf  Clrrk:  K.  li.  Jones*. 

Hiftrhrmir  Pivuiott:  M.  Dorset,  cliu*f;  Jam»«  A.  Kmerv,  acfiKtant  chief. 

linirii  Dhittian:  Ko.  II.  Wehmter,  chief;  C.  B.  Lan'k,  aiwiiitant  ctiief. 

JtuiM-dion  DiritUm:  Rick  P.  Steddom,  chief;  V.  (t.  HofCK,  anKxiate  rliief;  Morrih 

Wooden,  asHistatit  cliief. 
I\tth(tlf>giral  Dirininu:  JonN  K.  M0111.ER,  chief;  IIenry  J.  Wamhbi'rn,  awistant  chief. 
(imirtmtinr  IHrifion:  Kkiiakd  W.  Hickman,  chief. 
Dinnion  Iff '/ahAwpi:  B.  II.  Ransom,  chief. 

Hxjter'numt  Stntiau:  K.  C.  S4  iiKoEr)EK,  siijKTinten«lent;  W.  K.  Cotton,  aHHiHtant. 
Animal  II>i»fMinilmttii:  (iKORiiK  M.  Rt»MMKi,. 
Editor:  Jambh  M.  Pickens. 

biuciiemic  division. 
fluff:  .M.  Dorset. 
.ImiWan/  Chitf:  James  A.  Kmery.    . 
Ment-iiuqtedion  iMffoniturifK:  T.  M.  Price,  chemist  in  chaiye  of  central  lalH)ratory; 

A.  E.  Graham,   Ralph  Hoagland,  C.   II.  SwAMiEK,  William  B.  Smith,   K.  .\. 

ItoYER,  Clarence  T.  X.  Mar^h,   Phii.ii'  ("astle.man,    K.    II.   Incjersoll,    Peter 

Valaer,  BMiiHtiint  cheiiiistn  in  hranch  laboratories. 
Jlitff-rholrrn  Inrettigationn:  Chief  of  Division  in  char^-;    B.   M.   lioLToN  and  C.   X. 

McBryde,  Iwicteriologists;  W.  B.  Xiles,  inspector  in  char)^  of  fieM  experiments. 
PnuUry  Digestion  Experiments:  K.  W.  Brown,  assistant  chemist  in  charge. 
Invertigationn  of  Ihpf  and  Dixinfertantf:  James  A.   Kmery,  chemist;  C.   X.  McBryde, 

lia^'teriolojfist. 
J'rriKiration  of  TubermHn  nml  }fallein:  Chief  of  Division  in  charj^-;  A.  M.  West  niul 

II.  J.   Shore,  assistant  l>act«riologists;    W.   8.   Stamper  an<i    II.   S.    McAclry, 

assistants. 

2 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 
Washingtmi,  D.  C,  December  27 ,  1906. 
Sir:  I  have  the  honor  to  transmit  for  publication  the  accompanjang 
paper  entitled   "The  Bacteriolytic  Power  of  the  Blood   Serum   of 
Hogs,"  by  B.  M.  Bolton,  M.  D.,  of  the  Biochemic  Division  of  this 
Bureau. 

The  experiments  herein  described  were  carried  out  with  certain 
strains  of  the  hog-cholera  bacillus,  and  are  a  part  of  the  general 
investigations  concerning  hog  cholera  which  have  been  conducted  for 
some  years  by  the  Biochemic  Division.  The  subject  of  bacteriolysis 
has  claimed  much  attention  from  bacteriologists  in  recent  years,  and 
Doctor  Bolton's  work  is  believed  to  be  of  interest  on  account  of  the 
light  it  throws  upon  the  defensive  mechanism  of  the  animal  bod}^  in 
its  fight  against  infectious  diseases. 

I  recommend  that  the  paper  be  published  in  the  bulletin  series  of 
this  Bureau. 

Respectfully,  A.  D.  Melvin, 

Chief  of  Bureau. 
Hon.  James  Wilson, 

Secretai'y  of  Agriculture. 

3 


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CONTENTS. 


Page. 

Introduction 7 

Definition  of  sera  used  in  experiments 8 

Nature  of  the  substance  causing  destruction  of  bacteria 9 

Methods  used  in  drawing  blood,  and  description  of  cultures  employed 14 

The  bactericidal  i)Otency  of  serum  from  the  same  hog  at  different  times . .  15 

Modifications  which  occur  in  the  bactericidal  power  of  serum  upon  standing  .  19 
Comparison  of  the  bactericidal  potency  of  the  same  serum  for  different  strains 

of  bacteria 21 

The  potency  of  serum  from  arterial  blood  compared  with  that  from  venous 

blood - 22 

Effects  of  heat  upon  the  bactericidal  power  of  hog  serum 24 

Effects  of  injecting  cultures  of  B.  cholene  suis  upon  the  bacteriolytic  power  of 

hog  serum  for  this  organism 27 

Effects  of  dilution  upon  the  bacteriolytic  power  of  normal  and  immune  serum.  35 

Dilution  of  normal  serum 35 

Dilution  of  immune  serum 39 

Summary  of  the  dilution  experiments 45 

General  discussion 46 

Summary  of  the  conditions  affecting  bacteriolysis 57 

The  theory  of  complement  diversion 58 

Conclusions 60 

Bibliography 61 

5 


ILLUSTRATIONS. 


ftkge. 
Fm.  1.  Illiintrution  of  the  inechani}<n)  of  I jarteriolysiH  according  to  the  Ehrli<?h 

hyiHjthe«i8 11 

2.  diversion  of  coinpU'inent  in  utxitluted  immnne  8emm 13 

3.  Purtiul  iHti-teriolysis  atxl  partial  liivursion  of  (tiinpleinent  in  diluteil 

immune  Herum 13 

4.  Diversion  of  complement  in  immune  serum,  not  licate<l,  and  without 

tlie  addition  of  foreign  complement 51 

6 


THE  BACTERIOLYTIC  POWER  OF  THE  BLOOD  SERUM 

OF  HOGS. 


INTRODUCTION. 

Aside  from  a  few  preliminary  experiments  with  cultures  of  the 
typhoid  and  colon  ])acilli,  the  present  investigation  was  confined  to 
the  reactions  taking  place  between  three  strains  of  B.  choler'se,  suis^ 
and  normal  and  immune  serum'  of  hogs.. 

The  bactericidal  properties  of  the  blood  serum  and  other  fluids  of 
the  bod}'  have  of  recent  3'ears  attracted  a  great  deal  of  attention  from 
bacteriologists,  and  the  investigations  of  the  subject  have  led  to  very 
far-reaching  results  of  both  theoretical  and  practical  importance. 
Without  attempting  to  enumerate  in  full  the  different  properties 
which  blood  serum  has  been  found  to  possess  in  its  various  reactions 
with  ])acteria  and  their  products,  those  properties  which  bear  upon 
the  present  investigation  may  be  briefl}'  stated  as  follows: 

The  blood  serum  of  many  health}^  animals  when  drawn  into  test- 
tubes  has  the  power  of  destroying  a  larger  or  smaller  number  of 
bacteria  when  these  are  introduced  into  it.  Thus  large  numbers  of 
anthrax  bacilli  are  destroyed  by  rabbit's  blood  serum  in  test-tube 
experiments.  Typhoid,  colon,  and  Asiatic-cholera  bacteria  are  .also 
destroyed  by  the  blood  serum  of  rabbits. 

The  same  serum  is  not  equally  potent  for  different  bacteria,  and 
the  serum  from  different  animals  of  the  same  or  of  different  species 
varies  in  bactericidal  potency  for  the  same  organism.  The  chemical 
reaction  of  the  serum  seems  to  exert  some  effect  upon  this  quality  of 
the  serum.  The  more  alkaline  the  serum  the  more  potent  is  its  action 
apparently,  and  consequently  the  venous  blood  has  been  found  some- 
times to  furnish  more  potent  serum  than  the  arterial  blood  from  the 
same  animal." " 

The  injection  of  animals  with  the  products  of  growth  of  bacteria 
produces  different  effects  upon  the  properties  of  the  blood  serum,  de- 
pending upon  the  kind  of  bacteria  employed  for  injection.  The  injec- 
tion of  the  bacteria  of  the  group  to  which  B.  clwlei'x  suh^  B.  typhosus^ 
and  B.  coll  communis  belong  appears  sometimes  to  have  no  effect  upon 

«The  figure  references  refer  to  bibliography  at  end  of  bulletin. 


8  BA(TKRIOLYTIC    POWKR   OF    BLOOD   8RRUM    OF   BOOS. 

the  Ijacteriridul  |X)t<MU'y  of  the  scrum.  At  other  timet^  it  Heems  to 
increawo  tht»  iK)tciu'V,  hut  frr<jiK»ntIy  it  has  a  very  jKHJiiIiar  «'lfoct,  for 
in  some  cases  the  serum  after  the  iujiH'tion  of  the  Kiiimal  will  no  lonjfer 
kill  any  of  the  hactcria  of  the  kiml  used  to  inject  the  animal,  hut  on 
the  dilution  of  the  scrum  with  an  indifTercnt  fluid,  such  as  salt  solution, 
it  Iwcomes  stronjfly  haoterit-idal  for  the  hacteria  of  this  kind.  But 
this  sertun  In^haves  like  the  scrum  from  an  unin(M-ulat(Hi  animal  with 
other  hacteria  than  those  with  which  the  animal  is  inoculated.  Thus 
the  sc'rum  from  the  hltM)d  of  a  nihhit  injected  with  typhoid  Iwicilli  will 
often  fail  to  kill  the  typlioid  l)a<illi  unless  the  serum  is  j^reatly  diluted, 
hut  it  will  kill  just  as  many  Asiatic-cholera  spirilla  as  it  did  Iwfore  the 
animal  was  injected  with  the  typhoid  hacilli.*  The  |)eculiar  l)ehavior 
of  the  scrum  from  an  animal  injected  with  a  culture  of  Inicteria,  in  it« 
failure  to  kill  the  species  of  Itacteria  with  which  the  animal  is  injectiKl 
unless  it  is  diluted,  is  called  the  Ncisscr-\Vcchsl)ergf '*  phenomenon, 
after  the  two  invcstij^tors  who  tii^st  ohs«'rved  the  reaction.      "^ 

DEFINITION   OF   SERA    USED   IN    EXPERIMENTS. 

The  serum  ohtained  from  an  animal  which  has  l>een  injected  with 
Iwu'tcria  is  spoken  of  as  innnune  scrum  in  contradistinction  to  the  serum 
from  an  unino<ulated  animal,  which  is  called  normal  or  fresh  serum. 
Thus  serum  ohtained  from  a  hog  injected  with  B.  eholeriv,  suim  would 
Im'  known  as  Ji.  c/ioirne  xni^  innnune  hog  serum.  So  wherever  immune 
serum  is  mentioned  in  the  present  paper  it  is  to  be  understood  that  it 
refers  to  the  serum  ohtained  from  an  animal  which  has  received  at 
least  one  injection  of  bacteria,  but  not  necessarily  that  the  serum  has 
either  innnunizing  or  germicidal  properties.  On  the  other  band,  where 
normal  or  fresh  scrum  is  mentioned,  it  is  to  l>e  understood  to  refer  to 
serum  obtained  from  an  uninoculated  animal. 

Serum  loses  in  bactericidal  potency  on  standing  after  being  drawn 
from  the  animal;  and  the  higher  the  temperature  to  which  the  serum 
is  exposetl  the  more  rapid  the  loss  of  |)otency.  It  may  remain  potent 
for  several  days  or  for  even  a  week  or  more  in  the  refrigerator,  but  if 
kept  at  Innly  temperature  it  generally  loses  all  hictericidal  properties 
in  three  or  four  hours.  Heating  at  55  or  56^^  C.  for  ten  or  tifteen 
minutes  also  robs  the  serum  of  its  Iwu'tericidal  power,  or  rather  this 
treatment  of  innuune  serum  susp<'nds  it^  Imctericidal  power,  which  is 
restored  by  the  addition  of  a  small  amount  of  fresh  serum.  This  sus- 
pension of  Ijactericidal  power  by  heating  at  65'^  or  56^  C.  is  called 
inactivating  the  serum,  and  wherever  inactivat^^d  serum  is  mentioned 
it  is  to  l>e  understo<Mi  to  refer  to  serum  which  has  been  treated  in 
this  way.  Inactivated  serum  to  which  fresh  serum  is  added,  and  which 
has  had  its  Iwcteriolytic  properties  restored  in  this  way,  is  termed 
reactivated  serum. 


HOW    BACTERIA    ARE    DESTROYED.  V 

NATURE   OF  THE   SUBSTANCE   CAUSING   DESTRUCTION   OF  BACTERIA. 

In  regard  to  the  nature  of  the  substance  or  substances  in  the  senim 
which  cause  the  destruction  of  the  l)actcria  which  are  introduced,  there 
is  some  difference  of  opinion.  Some  authorities  maintain  with  Buch- 
ner  that  there  is  only  one  substance,  called  b}^  him  "alexin,"  wnich 
causes  bacteriolysis,  while  others — and  these  appear  to  be  in  the  major- 
ity— maintain  that  there  are  two  substances  concerned.  Those  who 
hold  to  the  former  view  regard  the  effect  produced  by  standing  or  by 
heating  as  due  to  the  w^eakening  of  the  alexin,  while  those  holding 
the  latter  view  explain  this  effect  by  a  modification  of  one  of  the  two 
substances  which  they  regard  as  necessary  for  bacteriolysis.  All 
are  not  agreed,  however,  as  to  the  nature  of  these  two  substances. 
Bordet  ^'^'^  and  the  French  school  generally  look  upon  the  substance 
which  is  not  modified  by  heat  as  a  sensitizing  agent  merely,  which  acts 
upon  the  bacteria  in  such  a  manner  as  to  make  them  susceptible  to 
the  destructive  action  of  the  other  body  to  which  the  bacteriolj^tic  prop- 
erties of  the  serum  are  directly  due.  Bordet  borrows  Buchner's  term 
"alexin"  for  this  active  agent,  and  he  gives  to  the  other  body  the  name 
"substance  sensibilisatrice."  According  to  the  French  school,  then, 
bactericidal  serum  owes  its  power  to  two  substances,  called,  respec- 
tively, substance  sensibilisatrice  and  alexin.  The  former  resists  heating 
up  to  75°  C.  or  even  somewhat  higher  temperatures  for  an  hour  or 
more,  while  the  latter  is  destro}  ed  by  heating  at  55°  C.  or  higher  in 
ten  or  fifteen  minutes.  Moreover,  the  substance  sensibilisatrice  accord- 
ing to  this  view  is  a  specific  substance  in  each  case.  The  substance 
sensibilisatrice  for  one  kind  of  bacterium  sensitizes  this  one  kind  only, 
and  while  it  can  not  of  itself  cause  bacteriolysis,  it  nevertheless  pro- 
duces certain  changes  in  the  bacteria.  On  the  other  hand,  according 
to  the  view  of  Ehrlich  and  his  school,  this  body,  the  thormostabile 
body,  causes  no  change  in  the  bacteria  themselves,  but  serves  merely 
as  an  intermediary,  a  binding  link,  serving  to  connect  the  bacteria  to 
the  active  body — the  alexin  of  Bordet — which  according  to  this  view 
is  the  active  agent  in  bacteriol3'sis.  To  the  two  hypothetical  bodies 
concerned  in  the  process  Ehrlich  has  given,  at  different  times,  differ- 
ent names.  At  first  he  called  the  thermostabile  body  ''intermediary 
body,"  afterwards  "immune  body,"  and  finally  "amboceptor."  The 
last  two  designations  are  those  now  generall}'  employed  by  the  Ehrlich 
school  for  the  body  which  corresponds,  as  is^  evident,  to  the  substance 
sensibilisatrice  of  Bordet.  To  the  other  body,  the  alexin  of  the  French 
school,  Elhrlich  has  given  variously  the  names  "addiment"  and  "com- 
plement," the  last  being  that  now  exclusivel}"  cmploj'ed.  The  term 
intermediary  body  has  been  emphned  in  a  recent  publication  for  the 
amboceptors  present  in  the  normal  serum  in  order  to  distinguish  these 
19500— No.  95—07 2 


10  BACTKRIOLYTir    POWER   OF    BLOOD   BKRUM    OF   H008. 

from  immuno  ainlKH-optorH,  or  ttiiilM>copt<)i*8  producwl  by  injecting 
jininmls  with  buctcria  or  their  productH. 

But  among  tho«e  who  have  acroptod  more  or  \vhh  completely  the 
Ehrlich  conception  of  the  nature  of  the  l)odieH  concerned  in  bacteri- 
olysis tiiere  is  difference  of  opinion  as  to  the  mo<ie  of  action  of 
the  complement.  Khrlich  himself  says  in  regaiil  to  the  matter 
"that  one  will  not  go  amiss  if  he  lissumes  with  Pfeiffer  that  the  proc- 
ess of  bacteriolysis  is  analogous  to  digestion,  and  attributes  to  the 
addiment  (complement)  thecharacter  of  a  digestive  ferment."  (irul)er,* 
on  the  contrary,  contends  that  the  complement  does  not  act  like  a 
ferment,  and  that  it  is  erroneous  to  draw  any  analogy  In'tween  the 
complement  and  an  enzym,  since  the  complement  is  entirely  used 
up  in  bacteriolysis,  whereas  in  the  process  of  fermentation,  as  is  well 
known,  the  ferment  is  not  used  up,  but  may  Ix?  recovere<l  after  the 
action  is  ended,  and  used  for  the  fermentation  of  other  material. 

Hut  whatever  the  exact  mode  of  action  ma}'  be,  it  is  evident  from 
what  has  just  Ijcen  said  that  both  the  Khrlich  and  the  Bordet  schools 
attribute  l)acteriol3'tic  action  proper  in  normal  serum  to  a  sul)stance 
easily  changed  b}'  comparativ<'ly  low  temperatures,  and  called,  respcc- 
tivel}^  complement  and  alexin  by  the  two  schools.  To  the  other 
body  concerned  in  bacteriolysis — the  amboceptor  of  Ehrlich  and  the 
substance  sensibilisatrice  of  liordet— is  assigned  by  the  former  the 
role  of  a  binding  link  between  the  complement  and  the  bacterium, 
while  by  the  latter  is  assigneti  to  it  the  property  of  a  sensitizer,  or  of 
a  mordant  as  in  dyeing.  In  the  one  case  the  bacterial  cell  is  regarded 
as  not  at  all  injured  or  otherwise  changed  b}'  the  union  with  the  aml>o- 
ceptor;  in  the  other  case  it  is  the  opinion  of  those  holding  this  view 
that  the  cell  is  acted  upon  and  changed  by  the  sensibilisatrice  in  such 
a  way  that  the  alexin  can  penetrate  it. 

Bordet*  summarizes  the  difference  Iwtween  his  theory  and  that  of 
Ehrlich  as  follows: 

According  to  Khrlich  and  Morgenroth  the  sixx-itic  antilxKly  (Hensibilisatrict?)  playp 
the  riMe  of  an  actual  int«rme<liary  (7.wiHchenkor|ti>r,  ainl>o<-e|>t'^>r),  a  link  of  union 
attaching  it^lf  on  tht>  one  haud  to  the  (*ell,  on  the  other  t4>  the  alexin.  In  other 
words,  the  alieorption  which  the  alexin  undergi>eH  in  the  pre>«n<t»  of  the  sentiitize<l 
cell  i«  not  due  to  an  affinity  nianift*j<ttHl  by  the  «vll  it(*elf  to  thin  Mil>ij>tam"e.  The 
alN<ori>tion  (»f  the  alexin  is  only  indin*ct;  the  «-ell  joins  itself  to  the  intermediary 
sulwtance,  which  in  itself  unite*!  chemically  by  its  other  i>ole  to  the  alexin. 

Our  idea  of  the  phenomenon,  which  we  feel  we  are  justified  in  holding,  in  alto- 
gether different  from  thit>.  To  im  it  neems  that  the  ttensibiliwtrice  which  unitei> 
with  the  cell  modifier  thlx  in  a  way  which  f»ermit«  it  to  abe«orb  the  alexin  dinnitly. 
The  action  of  the  wnsihilisatrice  upon  the  cells  is  comparable  to  that  of  certain  fixa- 
tive agent«  or  monlantH  which  confer  n|)on  certain  sulti'tanceft  (or  to  the  «-ells  of 
these  sulw<tanc(>s,  as  ii*  the  ca><e  in  histological  technic)  the  i>ower  of  alieorbing  colore 
which  they  refuse  to  al>sorb  before  treatment.  *  •  ♦  It  is  to  be  clearly  under- 
stood, however,  that  when  we  s|)eakof  morrlants  in  this  connection  we«lo  not  intend 
to  apply  in  all  details  the  phenomena  of  dyeing  to  the  matter  at  present  under  con- 
sideratioD ;  we  merely  mean  to  draw  a  comparison  which  will  serve  to  make  our 


HOW    BACTERIA    ARE    DESTROYED. 


11 


idea  clearer.  The  hypothesis  which  we  wish  to  bring  out  in  relief  is  that  in  the 
presence  of  hemolytic  serum,  the  cell  becomes  capable  of  absorbing  directly  the 
alexin  by  means  of  its  own  proper  elective  affinity,  and  that  this  power  is  due  to  a 
change  caused  by  the  sensibilisatrice.  In  other  words,  we  do  not  believe  that  one  is 
forced  to  admit,  with  Ehrlich  and  Morgenroth,  that  the  sensibilisatrice  itself  com- 
bines with  the  alexin,  and  that  this  union  is  indispensable  for  the  latter  substance  to 
attack  the  cell. 

Bordet  furthermore  states  in  the  same  connection  that  he  agrees 
with  Buchner  in  regarding  the  alexin  for  blood  cells  and  for  bacteria 
as  identical — that  one  and  the  same  alexin  may  attack  the  most  diverse 
cells;  whereas  Neisser  and  others  of  the  Ehrlich  school  believe  that 
alexins  or  complements  are  different  in  one  and  the  same  serum. 

While  it  is  evident  from  the  above  that  the  terms  amboceptor  and 
sensibilisatrice  are  used  to  designate  the  same  substance,  it  is  scarcely 
correct  to  use  them  interchangeably,  since  they  connote  somewhat  dif- 
ferent attributes  in  the  body  to  which  they  refer.  The  same  is  true 
of  the  terms  complement  and  alexin,  though  to  a  less  degree. 

The  following  diagrams,  obtained  from  various  sources  and  modified 
to  suit  the  purpose,  will  serve  to  illustrate  the  process  of  bacteriolysis 
according  to  the  views  of  the  Ehrlich 
school. 

Figure  1  represents  in  its  simplest 
form  the  mechanism  of  bacteriolysis 
according  to  the  Ehrlich  hypothesis, 
and  serves  to  illustrate  the  process  suffi- 
ciently'^ for  the  purposes  of  the  present 
paper. 

In  the  diagram  the  bacteria  are  rep- 
resented b}'  the  parts  marked  5,  the  am- 
boceptors b}'  those  marked  «,  and  the 
complements  b}^  those  marked  l'.  In 
No.  1  the  bacterium,  amboceptor,  and 
complement  are  lepresented  as  just  on 
the  point  of  uniting.  No.  2  represents 
the  bacterium  and  the  amboceptor  united  and  the  complement  on  the 
point  of  uniting  with  the  unoccupied  end  of  the  amboceptor.  No. 
3  represents  the  process  of  uniting  of  bacterium,  amboceptor,  and 
complement  completed;  the  bacterium  in  this  case  would  undergo 
bacteriolysis. 

It  should  be  borne  in  mind  that  according  to  this  theory^  bacterioly- 
sis can  take  place  only  where  the  bacterium  becomes  united  to  an 
amboceptor  which  is  itself  united  with  a  complement.  A  bacterium 
ma}'^  become  united  with  a  free  amboceptor — i.  e.,  an  amboceptor 
which  is  not  united  with  a  complement — but  the  bacterium  in  such  a 
case  does  not  undergo  bacteriolysis  unless  a  complement  subsequently 
becomes  attached  to  the  amboceptor.  The  complement  is  incapable  of 
uniting  directly  with  a  bacterium;  it  can  do  this  only  through  the 


Fig.  1. — Illustration  of  the  mechanism  of 
bacteriolysis  according  to  the  Ehrlich 
hypothesis. 


12  BACTKRIOLYTIC   POWKR   OK    BLOOD   SERI'M    OK   H0O8. 

intervention  of  the  aniiKH'cptor.  Hut  when  the  eompienient  J)econie8 
linl(C(l  to  tlie  Waeteriiun  l>v  means  of  the  jimhcM-eptor  the  ^mrteriunj 
iK^eomes  broken  up  into  minute'  ^nuiules  und  ultimately  disappearH. 

The  Ixjnds  by  which  the  amlxM-eptor  attaches  itself  to  the  iMU'teriuni 
on  the  one  hand,  and  to  the  complement  on  the  other,  are  ctillcd  hapto- 
pht)r  groups  or  haptophors  (/i),  and  similarly  this  name  is  given  to  the 
bonds  of  union  of  the  ))aeteria  and  of  the  complement.  The  ambo- 
ceptor thus  has  two  haptophors,  one  by  means  of  which  it  attaches 
it.s(»lf  to  the  bacterium,  the  cytophylic  haptophor,  and  one  by  meaiw 
of  which  it  attaches  itself  to  the  coniplement,  the  complementophylic 
haptophor.  The  bacteria  probably  |K)sses.s  each  many  haptophors  all 
of  the  same  kind — i.  e. ,  haptophors  ca{Mib]e  of  uniting  with  amboceptors 
of  the  same  kind  -l)ut  for  the  sake  of  simplicity  the  bacti'rium  is  repre- 
sented in  the  diagram  as  having  oidy  one  haptophor.  The  complement 
has  one  haptophor  group  and  one  so-called  toxophor  group  {()„  and  it 
is  by  means  of  the  latter  group  that  the  complement  a<'ts  up^m  the 
Imcterium.  The  complement  may  be  deprived  of  this  toxophor  group, 
and  although  it  is  still  cai)able  of  uniting  with  the  amlx)ceptor  in  such 
a  case,  it  can  no  longer  cause  Iwicteriolysis.  This  loss  of  the  toxophor 
group  is  caused  by  heating,  and  it  also  occurs  spontaneously  in  the 
serum  on  standing.  Bacteria  subjected  to  the  action  of  heated  serum 
do  not  undergo  Imcteriohsis,  but  become  fixed  to  the  an)l>oceptor8, 
and  the  amboceptors  become  united  to  the  haptophor  group  of  the 
complement  which  are  left  unaffected  by  the  heating.  It  will  thus  be 
readily  understood  why  bacteria  treated  with  heated  innnune  serum  are 
subsequently  protected  from  biu-teriolysis  even  when  unheated  immune 
serum  or  when  miheated  complement  is  added  to  them.  The  comple- 
mentophylii-  haptophor  of  the  aml>oceptor  is  in  such  a  case  already 
(X'cupied  by  the  haptophors  of  the  heated  complement,  which  ha8 
in  this  way  l)ecome  deprived  of  its  toxo])hor  group. 

The  amboceptors  ft)und  in  ordinary  normal  serum  are  either  all 
alike — and  in  this  case  they  nmst  possess  atKnity  for  a  great  many  dif- 
ferent kinds  of  bacteria  -or  they  nuist  differ  from  one  another;  and 
in  this  case  there  must  evidently  l)e  a  great  many  s|X'citic  amlH)ceptors, 
some  fitted  for  tlie  iwicteriolysis  of  one  species  of  ba<-terium,  some  for 
othei*s.  This  matter  seems  not  yet  to  have  !)een  s«'ttled.  But  it  is 
certain  that  the  injection  of  an  animal  with  certiiin  bacteria  or  their 
products  causes  the  formation  of  a  large  number  of  specific  am>K>- 
ceptors;  that  is  to  say,  of  amlxx'eptors  having  affinity  only  for  the 
kind  of  bai'teria  with  which  the  animal  is  injected.  Such  injections 
seem  not  to  increase  the  amount  of  complement. 

Complement  is  found  normally  in  the  serum,  that  of  some  animals 
|)08.sessing  more  than  that  of  others.  The  horse  apiM>nrs  to  have  a 
large  amount  of  complement  in  the  scrum.  It  is  not-  yet  settled 
whether  the  complement  Is  s|M'cific — that  is,  whether  the  complement 
for  one  kind  of  immune  serum  can  unite  with  the  amboceptors  of  thu 


HOW    BACTERIA    ARE    DESTROYED. 


13 


Fig.  2. — Diversion    of  complement   in  undiluted    immune 
serum. 


senim  onl\'  and  not  with  the  immune  serum  of  a  different  sort — or 
whether  tomplements  are  j^eneral;  thouj^h  they  seem  for  the  most 
part  not  to  be  specific.  The  complement  in  the  serum  of  iiorse's  blood 
seems  capable  of  reactivat- 
ing heated  immune  serum 
of  various  kinds.  Still  in 
some  cases  it  would  appear 
as  if  they  were  specific. 

With    the    explanation 
given  above  of  the  nature 
of  amboceptors  and  com- 
plements, the  phenomena 
which   take  place  in   im- 
mune serum  become  more 
or  less  satisfactoril}^   ex- 
plicable.    By  means  of  the  characteristics  ascribed  to  these  bodies  it 
is  possible  to  account  for  the  peculiar  behavior  of  imnuine  .^erum 
stated  above,  consisting  in  the  fact  that  such  serum   is  frequently 
more  potent  when  diluted  than  when  it  is  undiluted. 

Neisser  and  VVechsberg  were  the  first  to  observe*  this  phenomenon, 
and  the  theory  which  they  advance  to  explain  it  they  veiy  appropri- 
ately call  the  theory  of  the  diversion  of  complement.  As  the  name 
implies,  they  attribute  the  lack  of  bacteriolj'^sis  in  the  undiluted 
immune  serum  to  the  turning  aside  of  the  complement  from  the 
bacteria,  or  rather  from  the  amboceptors  which  are  attached  to  the 
bacteria.  They  hold  that  this  diversion  is  brought  about  by  the  free 
amboceptors  themselves.  In  other  words,  where  there  are  more 
amboceptors  than  there  are  complements  present  in  a  serum,  a  part  of 
these  attach  themselves  to  the  bacteria  and  a  part  to  the  complements. 

The  accompanying  diagram  (fig.  2), 
taken  from  Neisser  and  Wechsberg, 
and  modified  to  suit  the  present  descrip- 
tion, shows  two  amboceptors  «,  at- 
tached to  bacteria  J,  and  four  ambo- 
ceptors attached  to  complements  Jc. 
Bacteriolysis  is  not  possible  in  such  a 
condition,  because  the  complements 
have  been  diverted  from  the  ambo- 
ceptors which  are  attached  to  the  bac- 
teria. Bacterioh'sis  can  take  place 
only  when  the  complenient  l)ecomes 
attached  to  the  bacterium  through  the 
medium  of  the  amboceptor. 
Figure  3  is  meant  to  show  the  same  serum  diluted  with  an  equal 
amount  of  salt  solution.  In  this  case,  with  the  same  number  of 
bacteria  added,  it  is  evident  that  one-half  of  them  would  be  killed,  as 


Fig.  3. — Partial  bacteriolysis  and  partial 
diversion  of  complement  in  diluted  im- 
mune serum 


14  HA(TKRIOLVTlC    POWKR   OF    BLOoD   8RRUM    OF   HOGS. 

is  indicated  by  the  coiiibiiiutioii  l>ctwccn  iNictoriti,  aiiilMiceptor,  and 
<-(>iiiploim>iit  in  No.  I.  Tlu»  other  half  of  t\\v  Itactciiii  would  evi- 
dfntly  osca|)0,  tlu»  coniplonicnt  hoinj;  divertt'd  l»y  tin'  five  anil>o<'(>ptor, 
as  shown  by  Nos.  2  and  H. 

.Vlthoujrh  those  .statonnMits  in  rojjnrd  to  Itarteriolysisand  tin'  niochan- 
ism  probably  involv«'d  aro  by  no  means  exhaustive,  they  will,  |>»'rhap8, 
serve  the  purposes  of  the  jiresent  investigation  and  to  explain  the 
results  here  obtained.  It  will  Ih>  notice<l  that  ex|>erinients  have  been 
ma(h'  with  a  view  to  throwing  additional  lijjfht  ujMjn  those  phases  of 
bacteriolysis  already  nu'ntioiu'd,  as  they  wen*  observed  in  hoj^'s  bl(K)d 
serum,  and  it  wouhl  apjiear  that  the  results,  although  ditfering  in 
some  instances  from  those  of  other  observers,  are  nevertheless  sus- 
ceptible of  interpretation  in  hanuony  with  more  <»r  less  linnly  estab- 
lished hypotheses. 

METHODS  USED   IN   DRAWING  BLOOD,  AND   DESCHIPTION  OF 
CULTXJIIES  EMPLOYED. 

The  blood  was  dmwn  from  healthy  hogs,  or  from  hogs  injected  with 
cultures  of  //.  v/io/er;t  xhih^  subcutaneously  or  intravenously,  as  staU;d 
in  each  case,  at  various  lengths  of  time  before  the  dmwing.  It  was 
obtained  (1)  by  cutting  otT  a  piece  of  the  tail  and  allowing  the  blood  to 
flow  into  stcrilize<l  tubes  or  flasks.  (2)  by  bleeding  from  an  artery  in 
the  ear,  or  (3)  b3'  inserting  a  canula  into  the  carotid  artery  or  jugular 
vein.  In  some  cases  the  blood  was  drawn  from  the  carotid  and  from 
the  jugular  at  the  same  operation.  After  dmwing,  the  bkxHl  wius 
usually  placed  in  the  refrigeralor  to  allow  the  serum  to  separate,  but 
in  a  few  cases  it  was  used  immediat<'ly  after  drawing. 

All  the  hogs  used  in  the  ex|)eriments  were  in  a  perfectly  health}' 
condition  to  start  with,  and,  as  far  as  could  l>e  ascertained,  had  never 
l)een  sick.     They  weighed  from  80  to  40  p«>unds  each. 

The  cultures  employed  were  obtained  during  the  course  of  former 
ex|)«'riments.  One  of  them,  G.  P.  4692,  had  been  repeatedly  pa.ssed 
through  guinea  pigs;  another,  Crawford,  had  been  carried  ah»ng  for  a 
numl)er  of  years  on  artificial  media  without  jMissage  through  animals; 
the  third.  F.  20,  had  also  been  carrieti  along  for  sevenil  years  on  arti- 
flcial  media  without  animal  passage.  These  cidtures  presented  minor 
points  of  ditference  from  one  another,  but  they  were  all  quite  typical 
for  B.  cholfru'  tttttM.  All  of  them  give  characteristic  growths  u{>on 
artiti<*ial  culture  media  with  the  usual  fermentation  reactions  of  the 
sug?irs,  and  the  other  features  of  li.  rhol* n*-  kuIh^  both  macroscopic 
and  microscopic,  though  it  is  true  that  the  Crawford  strain  grew  more 
vigorously  and  gave  larger  an<l  denser  colonies  than  the  other  two, 
and  that  the  (J,  P.  4tJl>2  strain  gave  the  least  vigorous  growth  on  arti- 
ticial  media.  Also,  in  the  tests  which  were  made  to  determine  the 
point,  the  (».  P.  4692  strain  was  much  more  strongly  pathogenic  for 


BACTERICIDAL    POTENCY    OF    SERUM. 


15 


guinea  pigs,  rabbits,  and  bogs  than  the  other  two  strains.  The  Craw- 
ford strain  was  the  least  virulent  of  the  three.  So  the  virulence  of 
the  three  organisms  was  in  inverse  ratio  to  the  vigor  of  growth  upon 
artificial  culture  media.  The  culture  of  B.  coU  communk  was  obtained 
in  the  course  of  former  experiments  from  the  feces  of  a  normal  hog. 
It  showed  no  pathogenic  properties. 

THE  BACTERICIDAL  POTENCY  OF  SERUM  FROM  THE   SAME  HOG 
AT  DIFFERENT  TIMES. 

The  bactericidal  potency  of  the  serum  from  the  same  hog  on  differ- 
ent days  was  tested  by  bleeding  a  hog  at  intervals  of  a  few  days, 
drawing  off  the  serum  after  the  blood  had  stood  in  the  refrigerator 
for  twenty-four  hours  or  less,  and  distributing  it  into  test  tubes — 1  c.  c. 
into  each  tube — then  adding  a  definite,  measured  amount  of  a  suspen- 
sion in  salt  solution  of  the  bacteria  to  be  tested.  At  the  same  time  a 
tube  of  1  c.  c.  of  salt  solution  was  inoculated  with  the  same  measured 
amount  of  bacterial  suspension  and  plates  made  inmiediately  to  deter- 
mine the  number  of  bacteria  added.  Plates  were  made  from  the  serum 
tubes  on  the  following  day.  The  serum  from  4  hogs  was  used  in  this 
experiment,  the  blood  in  all  four  experiments  being  drawn  from  the 
tail.     The  results  will  be  found  in  the  following  tables: 

Table  I. — Bacteriolytic  action  of  normal  hoy  serum  from  the  same  animal  at  different 
drawings.     Blood  drawn  from  the  tail.     Hog  No.  17S3. 


Date. 


1906. 

April    7 

7 

9 

9 

11 

11 

14 

14 

19 

19 

22 

22 

26 

26 

May    3 

3 

6 

5 


Time  after  drawing. 


No.  of 
drawing 
of  blood.a 


3  hours . 

do.. 

do.. 

do.. 

do.. 

do.. 

.....do.. 
do.. 

2  days  . . 

i  day  . . . 
do.. 

3  hours . 

do. 

do.. 

do.. 


I 

I 

II 

II 

III 

III 

IV 

IV 

V 

V 

VI 

VI 

VII 

VII 

VIII 

VIII 

IX 

IX 


Number  of 
Culture  used  to  test    ]    bacteria 
bacteriolysis.  introduced 

per  1  c.  c. 


G.  P.  4692 
B.  coli  . . . 
G.  P.  4692 
B.  coli  . . . 
G.  P.  4692 
B.  coli  . . . 
G.  P.  4692 
B.  coli  . . . 
G.  P.  4692 
B.  cM  . . . 
G.  P. 4692 
B.  coli  . . . 
G.  P.  4692 
B.  coli  . . . 
G.  P.  4692 
B.  coli . . . 
G.  P.  4692 
B.  coli  . . . 


4,620 

5,880 

30,800 

7,000 

4,160 

225 

9,800 

9,520 

8,655 

2,600 

4,968 

1,940 

1,800 

2,800 

22,628 

28,380 

8 

869 


Number  of 

bacteria 

per  1  c.  0. 

after  1  day. 


79,800 

30,800 

48,300 

0 

9,800 

53 

4,690 

9,800 

142,900 

0 

43,200 

240 

1,220 

16,870 

17, 510 

84 

1,004 

2,594 


a  Roman  numerals  denote  the  serial  numbers  of  the  drawings  of  blood. 

As  will  be  seen  from  Table  I,  the  blood  was  drawn  from  hog  1733 
on  nine  difierent  days  and  tested  simultaneously  upon  cultures  of  G.  P. 
4692  and  on  the  culture  of  B.  coli  communis.  In  four  of  the  draw- 
ings the  serum  apparently  had  no  bacteriolytic  power  for  the  strain  of 
B.  cholerse  suis  employed,  while  in  four  of  them,  although  there  was 
also  no  marked  bacteriolysis,  there  was  nevertheless  no  evidence  of 


1« 


BACTKRIOLYTIC    1»<)WKR   OF    BLOOD   SERUM    OK   H0O8. 


any  jjreat  ItuTtnuse  of  th<»  bactoria  intrcKluccHl.  In  the  Honim  from  one 
of  the  dniwinjjM  the  Itactoria  were  rwluced  to  one-half  of  the  number 
intrixhiced;  this  was  the  jjn'atest  amount  of  potency  Hhown  in  any  of 
the  (Imwin^s. 

With  theeolon  ImciiluH  there  was  marked  ImcteriolyHis  in  onedraw- 
injf,  less  marked  in  four  other  drawings,  and  neither  increase  nor 
decrease  in  one  of  tiie  dniwinj^s,  w  hile  in  the  other  three  drawingH 
there  wa«  an  increase  of  the  bacteria  introduced. 

It  is  evident  that  the  serum  from  this  hojj  showed  different  degree^! 
of  |M)tency  or  absence  of  jxiU'iicy  ujion  the  different  occasions  when 
the  blood  was  dmwn. 


TablR  II. — li^icterioiiftir  iiclinn  of  uormnl  hoy  nerum  from  Oif  mme  animal  at  different 
drtiwinga.     Hloini  draim  from  the  tail.     IIwj  No.  1740. 


Date. 

Time  after  drawing. 

No.  of 

drawing 
of  blood. 

Ctiltun-  iimmI  tu  teat 
bactcriolyiiiti. 

Number  of 

bacteria 
introdured 
per  1  c.  c. 

Number  of 

bacteria 

per  1  c.  c. 

after  1  day. 

I9». 
Mar    « 

SboaiB 

I 
I 

II 
II 
III 
IV 
IV 
V 
VI 
VI 

O.P.  4692 

4 

975 

4.905 

8.778 

102 

1.010 

S.110 

848 

138 

1,700 

1.780 

do 

It.  roli 

8 

g 

do 

(i.  P.  4602 

i.sra 

70 

8 

do 

H.  coli  

9 

do 

O.  P.  4692 

l.ZM 

1.652 

9M 

10 

do 

«.  P.  4092 

10 

do 

h.  cfjli 

11 

do 

O.  P.  4692 

460 

12 

do 

G.  P.  4092 

0 

U 

do 

B.cM 

1,840 

The  blood  of  hog  1740,  as  is  shown  in  Table  II,  was  drawn  on  six 
different  days  and  tested  on  //.  ro/i  and  on  (i.  P.  4H92.  It  showed 
market!  Ijiictericidal  power  for  the  B.  coli  culture  in  three  of  the 
four  drawings  tested  with  this  organism,  but  in  one  there  was  no 
decrease  in  the  numl)er  of  Imcteria  introduced.  With  the  B.  cholene 
guix  culture  it  will  be  noticed  from  the  table  that  the  effect  varied 
greatly  with  the  different  drawings.  While  there  was  no  very  marked 
decrease  in  any  ca.se,  it  is  evident  in  general  that  the  bacteria  did  not 
multiply  abundantly  in  the  .serum  in  any  ca.se.  It  would  seem  as  if 
growth  had  lieen  inhibited  by  the  .serum,  although  active  Iwicteriolysis 
was  lacking  except  in  the  serum  from  one  drawing. 

Table  III. — lUnieriolytic  action  of  normal  hog  nertim  from  the  aame  animal  at  different 
drairingn.     Blood  draim  from  the  tail.     Hog  So.  1741. 


Dale 


Time  after  drawing. 


1906. 
May     H     3  hnun 

8   do. 

9   do. 

9  i do. 

10  do. 

10  do. 

11    do. 

12  , do. 


No.  cif 
drawing 
o(  blood. 


I 
I 

II 

11 

III 

III 

IV 

V 


Culture  uaed  to  teat 
bacteriolyrfa. 


G.  P.  4092 
B.eoU... 
U.  P. 4092 
H.tvti... 
a.  P.4fl«2 
H.rnti... 
a.  V.  4092 
B.eoU... 


Number  of  Number  of 

bacteria       bacteria 
Introduced    per  1  c.  c. 
per  1  c. c.    aftcrldajr. 


4.996 
8,778 

103 
1.990 
1.010 
S.1M 

848 
1.700 


tt 

14 

108 

990 

1.912 

40 

SO 

0 


BACTERICIDAL  POTENCY  OF  SERUM. 


17 


As  Table  III  shows,  the  blood  was  drawn  from  hojj;'  1741  on  five 
different  occasions.  The  serum  from  four  of  the  drawinj^s  was  tested 
upon  the  culture  of  B.  coli^  and  it  showed  marked  bacteriolytic  prop- 
erties for  this  organism  in  all  the  tests.  The  serum  from  four  of  the 
drawings  was  tested  upon  the  G.  P.  4092  strain  of  B.  cholerae  suix^  but 
onh**  two  of  the  drawings  showed  bacteriolytic  properties  for  this 
organism. 

Table  IV. — Bacteriolytic  action  of  normal  hog  serum  from  the  mme  animal  at  different 
drawings.     Blood  drawn  from  the  tail.    Hog  No.  174^. 


Date. 


i9a5. 

Vav  8 
8 
10 
10 
12 
12 
11 
13 
23 
23 
23 
24 
24 
24 
26 
26 
29 
29 
29 
June  5 
5 
5 


Time  after  drawing. 


3  hours 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
....do. 
^  hour . 
....do. 
....do. 


No.  of 
drawing 
of  blood. 


I 

I 

II 

II 

III 

III 

.    IV 

V 

VI 

VI 

VI 

VII 

VII 

VII 

VIII 

VIII 

IX 

IX 

IX 

X 

X 

X 


Culture  used  to  test 
bacteriolysis. 


G.  P.  4692 
B.  coll  .... 
G.  P.  4692 
B.  coli .... 
G.  P.  4692 

B.  coli 

G.  P.  4692 

B.  colL 

G.  P.  4692 
Crawford 

F.  26 

G.  P.  4692 
Crawford 

F.  26 

Crawford 
F.26 

F.  26 

Crawford 

G.  P.  4692 

F.26 

Crawford 
G.  P.  4692 


Number  of 
bacteria  in- 
troduced 
per  1  c.c. 


4,925 

3,788 

102 

1,970 

1,010 

3,110 

848 

1,700 

10,880 

40,500 

19,040 

1,682 

1,808 

12,040 

320 

80 

1,740 

2,320 

1,920 

2,840 

3,380 

1,380 


Number  of 
bacteria 
per  1  c  c. 

after  1  day. 


82 

686 

46 

82 

798 

30 

50 

0 

3,376 

2,640 

18,280 

0 

180 

1,640 

0 

1,960 

4,480 

0 

%0 

26,640 

180 

4,420 


In  the  experiments  with  the  blood  of  hog  1742,  as  shown  in  Table 
IV,  ten  different  drawings  were  made  on  separate  days,  and  four  of 
them  tested  on  the  culture  of  the  colon  bacillus,  eight  on  culture  G.  P. 
461)2,  live  on  culture  Crawford,  and  five  on  culture  F.  26. 

The  colon  bacillus  was  greatly  diminished  in  all  cases.  In  one  case 
where  1,700  per  1  c.  c.  had  been  introduced  into  the  serum  all  of  them 
were  destroyed,  and  in  another  test  3,000  out  of  4,000,  in  round  num- 
bers, were  killed. 

The  tests  with  the  G.  P.  4692  culture  showed  that  the  serum  was 
bactericidal  in  seven  tests  out  of  eight.  In  one  of  these  about  1,700 
bacilli  were  introduced  and  all  of  them  were  killed.  On  the  other 
hand,  in  the  serum  from  one  of  the  drawings  there  was  an  increase  of 
the  bacilli  introduced. 

The  tests  with  the  Crawford  culture  showed  that  the  serum  from  all 
of  the  drawings  tested  was  actively  bactericidal,  but  in  some  more 
than  in  others.  In  one  of  the  drawings  about  38,000  bacilli  per  1  c.  c. 
of  serum  were  destroyed  out  of  the  40,000  introduced.  But  in  another 
case  all  were  not  destroyed  although  only  1,808  were  introduced. 
19500— JSTo.  95-07 3 


18  BACTERIOLYTIC    POWER   OK    BLOOD   SERl'M    OF    H(X}8. 

Testi*  with  culture  F.  26  nbowod  liactoriolysiH  in  one  drawing;;  in 
another  drawinjf  there  wa.s  no  increase  nor  decrease  of  the  bacteria 
introduced.  In  three  drawingH  there  was  marked  incroat»e  of  the 
oiyanisnit*  intriKluccd. 

That  the  bactericidal  potency  of  the  serum  of  hogs  nliould  vary  at 
different  timcM,  as  these  results  seem  to  indicate,  should  |)erluii)s*not 
1h»  u  mutter  of  surprist^  There  are  probably  many  circiunstances 
which  influence  this  proj)erty  of  the  scrum.  As  i«  stated  elsewhere  in 
this  pa|)cr,  the  chemical  reaction  of  the  serum  ha.s  l)een  found  by 
others  to  inHucnce  the  l)uctericidal  |K)wer,  and  doubtless  there  are 
other  a.s  yet  obscure  circumstances  which  raise  or  lower  this  power  of 
the  serum.  The  nature  of  bacteriolysins  will  Ih»  found  (lis<'ussed  at 
some  lenjfth  Inflow  in  a  different  connection,  and  it  would  not  seem 
at  all  improbable  that  there  ma>'  be  more  of  these  at  one  time  than 
at  another  present  in  the  l)lood  serum,  Imleed,  the  pHnluction  of 
iMicteriolysis  in  serum  of  the  living  animal  seems  to  l>e  easily  intluenced 
one  way  or  another,  and  it  would  not  be  unreasonable  to  regard  them 
as  varying  from  time  to  time  under  even  slightly  changing  conditions 
of  the  body. 

Tronnnsdorf  "•  noticed  that  human  sera  derived  from  normal  indi- 
viduals as  well  as  from  those  suffering  frou)  various  diseases  vary 
greatly  in  bacteriolytic  jwwer.  Petterson'*  found  the  same  thing 
with  chickens. 

Morgenroth  and  Sachs"  found  great  variation  in  cytolytic  power  in 
serum  of  various  sorts.  Thus  the  serum  from  a  horse  at  one  drawing 
was  hemolytic  for  rabbits'  corpuscles  but  not  for  those  of  guinea  pigs; 
three  days  later  the  serum  from  the  same  horse  was  strongly  hemo- 
lytic for  guinea  pigs'  corpuscles,  but  only  very  slightly  for  rabbits' 
corpuscles;  twenty-three  days  later  the  serum  from  this  horse  was  not 
hemolytic  for  guinea  pigs'  coipuscles,  but  strongi}'  hemolytic  for 
rabbits'  corpuscles. 

It  is  therefore  evident  that  the  cytolytic  power  of  serum  is  very 
variable.  Not  only  does  the  blood  from  different  individuals  of  the 
same  species  differ  in  this  respect,  but  the  serum  from  the  same  indi- 
vidual differs  from  time  to  time.  This  is  prolmbly  the  case  with  all 
animals,  and,  as  is  apparent  in  the  experiments  with  hogs,  these  are 
no  exception  to  the  rule. 

In  regard  to  the  above  experiments,  while  there  is  more  or  less 
variation  b<»tween  single  tests,  if  one  strain  only  of  B.  rhohnf  mth'm  is 
considered,  there  seems  to  be  a  general  tendency  toward  either  active 
bacteriolysis  or  else  inhibition  of  growth. 


MODIFICATIONS    IN    BACTERICIDAL    POWER    OF    SERUM. 


19 


MODIFICATIONS  WHICH   OCCUR   IN   THE   BACTERICIDAL   POWER 
OF  SERUM  UPON  STANDING. 

As  has  been  previously  stated,  the  blood  was  placed  on  ice  in  all 
cases  where  it  was  not  at  once  used  for  making  tests.  In  some  cases 
the  bactericidal  properties  of  the  serum  were  tested  after  the  serum 
had  stood  for  various  lengths  of  time.  The  results  of  these  tests,  as 
will  be  seen  from  the  accompanying  tables,  show  in  general  that  the 
bactericidal  potency  is  retained  in  some  cases  for  as  long  as  nine  days. 
Occasionally,  however,  the  bactericidal  power  of  the  serum  is  dimin- 
ished by  even  two  days'  standing. 

Table  V. — Bacteriolytic  action  of  normal  hog  serum,  after  standing  for  various  lengths  of 
time  in  the  refrigerator.     Blood  drawn  from  the  tail,  eighth  drawing.     Hog  No.  1742. 


Number  of '  Number  of 

Date. 

Time  after  drawing. 

Culture  used  to  test 
b.icteriolvsis. 

\    bacteria    j    bacteria 
introduced    per  1  c.  c. 

per  1  c.  c. 

after  1  day. 

1905. 

V 

1 

Mav   2f. 

3  hours 

Crawford 

320 

0 

June    2 

7  dav.s 

do 

1,%0 

380 

May  26 

3  hours 

F.  26 "l. 

1                 80 

1,960 

June    2 

7  days 

do 

j           3,280 

2,980 

The  serum  from  the  blood  of  hog  1742  at  the  eighth  drawing,  as  is 
shown  in  Table  V,  was  tested  upon  two  of  the  organisms  only, 
namely,  Crawford  and  F.  26.  For  the  Crawford  culture  the  bacteri- 
cidal power  of  the  serum  was  retained  for  seven  days,  whereas  for  the 
F.  26  culture  the  serum  was  bactericidal  at  the  start  and  only  inhibi- 
tory after  seven  days. 

Table  VI. — Bacteriolytic  action  of  normal  hog  serum  after  standing  for  various  lengths  of 
time  in  the  refrigerator.     Blood  drawn  from  the  tail,  ninth  drawing.     Hog  No.  17 4^. 


Date. 

Time  after  drawing. 

Culture  used  to  test 
bacteriolysis. 

Numberof 

bacteria 
introduced 
per  1  c.  c. 

Number  of 

bacteria 

per  1  c.  c. 

after  1  day. 

1905. 
May  29 
June    3 
May  29 
June    3 

3  hours 

F.  26 

1,740 
1,220 
2,320 
1,060 
1,920 
700 

4,480 
1  180 

5  days 

do. 

0 

do 

0 

Mav  29 

3  hours 

G.  P.  4692 

960 

do 

1,100 

At  the  ninth  drawing  from  hog  1742,  as  Table  VI  shows,  the  serum 
retained  its  bactericidal  potency  for  the  Crawford  culture  apparently 
unabated  for  five  days,  whereas  for  the  other  two  organisms  there  was 
apparently  more  or  less  inhibition  of  growth,  perhaps,  both  at  the  start 
and  after  five  days,  but  no  very  marked  bacteriolysis  for  either  of 
them. 


20 


HACTKRIOLYTIC    POWKR    OF    BLOOD    SKKL'M    OF    H008. 


Table  VII. — HtiHrriolytic  action  o/  tutrmal  ht*g iierum  after  tUmding/ur  variuun  tettglha  oj 
time  in  the  rffrigertUor.     lilottd  drxncn  from  the  tail,  tenth  drttwing.     Iltig  Xo.  17 4f. 


IlfttO. 


June   {*    4  hour.... 


riin«?  MfUr  drawing. 


6  ld«y. 

7  .  Zdaya.. 
Hdayii.. 
4  day* . . 
Mdajm. 

{hour., 
day... 
2  days.. 
Sdayn.. 
4  day*.. 
14  days, 
t  hour. . 
Iday... 

2  days. 

3  days. 

4  days. 


19    14  days. 


Culture  used  to  teat 
bacteriolyata. 


NnmbiTof  Number  (if 

bacti'ria  '  iNU-teria 
Introduced  uerlc.e. 
I  per  I  c.c.    after  I  day. 


F.  26. . 

do.... 

do.... 

do.... 

do.... 

do.... 

Crawford . 

do.... 

do... 

do.... 

do.... 

do.... 

O.  P.  4092. 

do.... 

do.... 

drt.... 

do.... 

do.... 


2.  MO 
2.240 
S,440 
Z200 

i.aoo 

4,040 

3.  aw 

7.440 

t.taa 

2,  MO 
1,440 
1.380 
3,040 
IW 

aso 

1.880 
1.740 


211.  MO 

M.TOO 

900 

1.S40 

1H.2<W 

a  ■» 

IM 

120 

7«> 

3.160 

1.3W 

» 

4,420 

6,320 

8,420 

2,000 


oThis  character  signiflcs  that  there  were  too  many  colonies  to  count. 

The  .»<rruni  from  the  blood  at  the  tenth  di-awinjf  from  hog  1742,  as 
Table  VII  shows,  preservod  it**  bactoricidal  power  to  a  marked  degree 
for  the  Crawford  culture  up  to  and  inchiding  the  .second  day  of  stand- 
ing. It  appears  to  have  been  somewhat  more  jK)tent  on  the  fourth 
than  on  the  third  day  of  .standing,  but  the  difference  in  i^otency  of  the 
serum  on  these  two  days  is  hardly  sufficient,  perhaps,  to  be  of  any 
great  significance.  F'or  tlie  other  two  strains  there  was  at  most  inhi- 
bition shown  in  .some  of  the  te.st.s,  but  no  decided  bacteriolysis  either 
before  or  after  stsmding,  except  perhaps  in  the  ca.se  of  the  F.  'J^J  strain 
after  two  days.  But  in  the  case  of  this  organism  the  serum  appeared 
to  have  Iwen  more  jwtent  after  standing  for  three  daj's  than  it  was 
previous  to  this  time. 

Tablk  VIII. — Jifideriolijlic  action  of  normal  hmj  sentm  after  standing  for  rarious  length* 
of  time  in  the  refriyertitor.  Blood  dratcn  from  the  tail,  eleventh  draving.  lUtg  So. 
174^. 


iMe 


190ft. 
)unc  12 
16 
19 
22 
12 
16 
19 
22 
12 
19 
22 


i  hour  . 
4  darn.. 
7  days. . 
10  days. 
)  hour  . 
4  days. . 
7  days. . 
10  days, 
i  hour  . 
7  days.. 
10  da)-s. 


Time  after  drawinir. 


■Number  of 
Culture  u^ed  to  test       txicteria 
baclcri()ly»i!(.  intmduri'd 

per  1  c.  c. 


F.26 

do.... 

do.... 

do.... 

Crawford. 

do.... 

do.... 

do.... 

G.  P.  4692 . 

do.... 

do.... 


2.aM0 

1,000 

4.040 

26.000 

2.U0 

X.MO 

1,440 

M.400 

16.120 

1.740 

8,880 


Number  of 
bacteria 
p»'r  1  c.  r. 

after  Iday. 


650 

1.180 

16.  .100 

■  as 
400 
280 
320 
«> 
800 
1.280 


■This  character  si^niAes  that  there  were  too  many  colonies  to  count. 


COMPARISON    OF    BACTERICIDAL    POTENCY    OF    SERUM. 


21 


The  serum  from  the  blood  of  hog  1742  at  the  eleventh  drawing,  as 
shown  in  Table  VIII,  had  marked  bactericidal  power  for  all  three 
strains  at  the  start,  and  this  was  retained  for  as  long  as  seven  days  for 
the  Crawford  culture,  whereas  there  was  inhibition  only  for  F.  26 
after  four  days,  and  after  this  there  was  not  even  inhibition  for  this 
organism.  The  effect  upon  the  G.  P.  4692  culture  was  similar  to  that 
upon  the  F.  26  strain. 

Table  IX. — Bacteriolytic  action  of  normal  hog  serum  after  standing  for  lurknis  lengths 
of  time  in  the  refrigerator.     Blood  dravm  from  the  jugular  vein,  twelfth  drawing.     Hog 

No.  1742. 


Date. 

Time  after  drawing. 

Culture  u.sed  to  test 
bacteriolysis. 

Number  of 
bacteria 

introduced 
per  1  c.c. 

Number  of 
bacteria 
per  1  c.c. 

after  1  day. 

1905. 
June  22 

J  hour 

Crawford 

4,320 
4,060 
3,380 

640 
1,240 

340 

23 

1  day 

do 

22 

i  hour 

F.  26 

23 

1  dav 

do 

22 

i  hour 

G.P.4692 

do 

23 

1  day 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 

The  serum  from  the  blood  of  hog  1742  at  the  twelfth  drawing  (Table 
IX)  was  taken  from  the  jugular  vein,  and  it  will  be  observed  from 
the  table  that  the  serum  showed  no  bacteriolytic  power  either  at  the 
start  or  on  standing.  In  order  to  test  whether  the  lack  of  bactericidal 
power  was  due  in  this  case  to  the  fact  that  the  blood  was  drawn  from 
the  vein,  experiments,  which  are  hereinafter  given  (page  22),  were 
made  to  compare  the  bactericidal  power  of  arterial  blood  serum  with 
that  of  venous  blood  serum. 


COMPARISON    OF    THE    BACTERICIDAL   POTENCY   OF   THE 
SERUM  FOR  DIFFERENT  STRAINS  OF  BACTERIA. 


SAME 


If  in  the  foregoing  tables  comparison  is  made  between  the  results 
obtained  with  the  same  drawing  of  blood  upon  the  various  organisms, 
it  will  be  seen  that  quite  decided  differences  appear. 

Selecting  the  most  striking  contrasts,  in  one  case  there  was  a  decided 
increase  of  the  B.  cholerse  suis^  culture  G.  P.  4692,  while  in  the  same 
blood  7,000  B.  coli  per  1  c.  c.  were  all  destroyed.  It  is  true  that  in 
this  case  a  great  man\'^  more  of  B.  cholet'ai  suis  were  introduced  at  the 
start  than  of  B.  coli^  and  this  may  have  influenced  the  result  some- 
what. But  in  another  case  where  approximately  the  same  number  of 
organisms  of  the  two  kinds  were  introduced  at  the  start  there  were 
very  few  of  the  B.  cholerse  suis,  if  any,  destroyed,  whereas  nearly  all 
of  the  B.  coli  were  destroyed.  In  still  another  case  there  was  a  very 
great  increase  of  B.  cholerae,  suis^  while  all  of  the  B.  colt  were  de- 
stroyed. In  one  case  there  was  no  notable  increase  of  the  B.  cholerse, 
suis,  while  there  was  a  decrease  of  the  B.  coli. 


22 


BACTERIOLYTIC    POWKR   OF    BLOOD   BERUM    OF   H008. 


It  is  evidont,  thercfuro,  that  the  bactericidal  i)ower  of  ho)f  8enim  is 
ditTereiit  for  this  strain  of  Ji.  choUrm  suin  tumX  for  the  B.  coliy  in  so 
far  as  this  can  be  dotcrniined  by  the  methods  at  present  in  use. 

THE   POTENCY   OF    SERUM   FROM   ARTERIAL    BLOOD    COMPARED 
WITH  THAT  FROM  VENOUS  BLOOD. 

As  heroinl>of()ro  statrd,  the  followinj;  oxporim«'ut.s  \\or«»  undertaken 
because  of  the  results  obtained  with  the  serum  from  the  venous  blood 
in  the  experiment  in  which  it  appeared  that  there  was  neither  inhibi- 
tory nor  Imctericidnl  jK)wer  in  the  serum.  In  the  experiments  pre- 
vious to  this  one  the  blood  was  dniwn  from  the  tail,  and  was 
consequently  mainly  arterial.  It  spurted  from  a  severed  artery  when 
the  tail  was  cut.  In  the  experiments  at  present  under  ronsideration 
the  blood  was  dnuvii  from  the  carotid  artery  or  from  the  ju^>-uIhi-  vein 
either  at  the  same  operation  or  on  different  occasions. 

Table  X. — Bacteriolytic  action  of  arterial  $erum  compared  with  that  of  vettous  terum, 
fmth  tern  from  the  mme  normal  hmj  (No.  17SS)  at  tlte  tame  operation.  Tenth  drawing 
of  Ittofxt.      7t  */j»  made  ai  intervaU  after  drawing. 


Time  after  drawing. 


Culture  used  to  tent 
bavterioljrijD. 


1  day P.M. 

2  dny » I do  , 

V  clays i do. 


1  dav. 

2  dayH. 
9dayit. 
Iday.. 
2davH. 
9  days. 


Crawford . 

do.... 

do.... 

O.  P.  40B2. 

do.... 

.....do.... 


Number  of 
tmctt-ria 

iniriMlueed 
per  1  c.  e. 


Nomber  of  bacteria 

rrr  I  c.  c  after 
day. 


Arterial 
•eram. 


Venoiu 
ncrum. 


8.880 

ao 

4,«20 

16.830 

880 

■i.aoo 

940 

40 

1,480 

200 

420 

140 

2,440 

9.700 

9.880 

8.100 

100 

100 

820 

aoo 

400 
80 
120 
40 
5,920 
6.680 
140 


The  serum  from  hog  1783  at  the  tenth  drawing,  as  shown  in  Table 
X,  was  taken  at  the  .same  operation  from  the  carotid  artery  and  from 
the  jugular  vein.  In  the  tests  with  the  Crawford  culture  the  serum 
from  the  art<>rial,  as  well  as  that  from  the  venous  blood,  seem  both  to 
have  posses.sed  <*onsiderable  j>oteney,  and  to  have  posse^ssed  it  in  alxuit 
equal  degree.  They  furthermore  api)eur  to  have  retained  their 
potency,  much  or  all  of  it,  for  nine  days.  For  G.  P.  4(>S>2  culture 
neither  .serum  ap|K»ars  to  have  had  strong  bactericidal  power  in  any 
cas<'.  They  lK)th  seem  to  have  had  .some  inhibitory  {wwer  for  this 
organism  both  at  the  start  and  after  nine  days.  For  culture  F.  26 
there  apix?ars  to  have  l»een  no  difference  between  the  two  kinds  of 
serum  after  having  .stood  for  one  day,  but  the  serum  from  the  venous 
blo<Kl  would  .seem  to  have  retained  its  potency  upon  standing  more 
tenaoiouslv  than  that  from  the  arterial  blood. 


AETERIAL    AND    VENOUS    BLOOD    SERUM    COMPARED. 


23 


Table  XI. — Bacteriolytic  action  of  arterial  strum  compared  with  that  of  venous  serum, 
both  sera  fr(»n  the  same  normal  hog  {N^o.  1733)  at  the  same  operation.  Eleretith  draw- 
ing of  blood.     Tents  m,ade  at  intervals  after  draiinng. 


Time  after  drawing. 


Culture  used  to  test 
bacteriolysis. 


1  day F.  26... 

2  days ' ' do  . 

5  day.s I do  . 

8  days do . 


Number  of 
bacteria 

introduced 
per  1 0.  c. 


1  day. 

2  days . 
f)  days . 
8  days. 
Iday.. 
2  days. 
5  days. 
8  days . 


Crawford . 

do.... 

do.... 

do.... 

G. P.  4692.. 

do.... 

do.... 

do.... 


5,420 

6,  .520 

8,580 

240 

8,160 

8,660 

26,240 

1,920 

2,160 

2,720 

a  00 

2,240 


Numberof  bacteria 

riT  1  c.  c.  after 
day. 


Arterial    Venous 
serum.      serum. 


10,480 

20,160 

280 

10,960 

160 

400 

20,640 

7,280 

2, 520 

2,960 

21,240 


220 

27,680 

8,960 

2,600 

10,860 

360 

580 

60 

3,300 

3,340 

1,360 

2,320 


a  This  character  signifies  that  there  were  too  many  colonies  to  count. 

The  blood  obtained  at  the  eleventh  drawing  from  hog  1733,  as  shown 
in  Table  XI,  was  drawn  from  the  artery  and  from  the  vein  at  the 
same  operation.  There  seems  to  have  been  a  dilEference  in  the  potency 
between  the  two  kinds  of  serum  for  all  three  organisms.  For  the 
Crawford  culture  the  serum  from  the  arterial  blood  seems  to  have 
been  quite  strongly  bactericidal  at  the  start,  but  the  serum  from  the 
venous  blood  was  apparently  not  so.  The  tests  made  after  the  serum 
had  stood  for  two  days  would  indicate  that  both  kinds  of  serum  were 
potent.  The  serum  from  the  venous  blood  in  this  case  seems  to  have 
gained  in  potency  upon  standing,  a  result  previously  noted  in  the 
case  of  culture  F.  26  with  a  different  serum,  but  it  seems  necessarj^  to 
conclude  that  an  error  of  some  kind  must  have  crept  in  where  such 
results  are  obtained,  since  they  are  at  variance  not  only  with  the 
a  priori  probabilities  of  the  case  but  also  with  the  usual  experience. 
On  the  whole,  the  results  in  this  experiment  seem  to  indicate  for  the 
Ci'awford  culture  a  greater  potency,  or  at  least  a  retention  of  a  greater 
amount  of  potency,  on  the  part  of  the  venous  than  of  the  aiterial  blood. 

For  culture  F.  26  in  this  experiment  the  arterial  serum  appeared  less 
potent  at  the  start  and  after  eight  days  than  the  venous  serum,  but 
after  standing  five  days  it  was  more  potent  than  the  venous  serum. 
Here  again  the  serum  seems  to  have  gained  potency  on  standing. 

For  G.  P.  4692  the  stronger  retention  of  potency  of  the  venous 
serum  as  compared  with  the  arterial  is  apparent  in  the  tests  made  five 
days  and  eight  daj^s  after  drawing.  No  difference  between  the  two 
sorts  of  serum  is  noticeable  in  the  tests  made  with  this  organism  one 
day  and  two  days  after  drawing. 

The  fact  that  blood  through  which  a  stream  of  COg  was  conducted 
becomes  more  alkaline  led  Hamburger'  to  make  tests  of  the  compara- 
tive bactericidal  potency  of  serum  from  ordinary  blood  and  that  from 


24 


BACTERIOLYTIC    l»OWKR    OF    BLOOD    flKRrM    OK    H<K»8. 


l)loo(l  throu);li  which  CO,  hml  lMM>n  iMissod,  an  well  &s  of  the  difference 
in  this  n*s|MM't  In'tween  artorinl  uiul  venouH  blood,  lie  found,  in  fiict, 
that  st'i'iini  from  venous  I>I(m>(1  and  from  blood  through  which  CO,  had 
Ikmmi  jMiMsrd  is  more  |)ot(Mit  than  art4'riaI-l>l(MHi  si-rum. 


EFFECTS  OF    HEAT   T7P0N  THE   BACTERICIDAL   POWEB    OF  HOG 

SERUM. 

A  larjfo  number  of  tosts  were  matle  paniUol  with  those  above  recorded 
to  determine  the  effect  of  heat  ujHjn  the  j^enniridal  |k)wim-  of  hog 
s<»rum,  and  also  to  det^'rmine  whether  there  is  any  difference  lietween 
the  |K)toncy  of  the  hejiU'd  serum  for  the  different  strains  of  li.  cholerie 
»uiji  upon  which  it  wa.s  tested. 

The  meth(xl  employed  in  making  the  tests  consisted  in  immersing 
the  tulws  (*ontaining  the  serum  up  to  the  plugs  in  a  water  l>ath  which 
was  kept  by  means  of  a  thermoregulator  exactly  at  the  temperature  to 
Ik?  tested.  The  results  are  shown  in  tlie  following  tables,  from  which 
it  will  be  seen  that  the  serum  was  subjected  to  temperatures  of  51^, 
62°,  5Z^^  and  64°  C.  for  ten  minutes  and  for  thirty  minutes  at  different 
times.  Tests  were  also  made  at  temperatures  above  54*-^  C  but  these 
are  not  tabulated  for  the  reason  that  they  gave  results  similar  to  those 
at  54°. 

Table  XII. — Effects  of  heat  H/w>n  the  bwlerioh/tir  jumer  of  normal  hug  terum.  Blood 
drawn  from  the  tail  and  kept  in  the  refrigeralor  for  varimin  lengths  of  time.  Temper- 
ature employed,  51"  C. 


Timo  ntlvr 
drawing. 

Number  of 
Iwcteria 

intn>dured 
per  1  c.  c. 

Arterial  blood. 

Date. 

No.  of  hog          Culturt'  ust-d 
and  drawing              to  ti-st 
of  blood.            liac'torlolydis. 

Number  of  bacteria 
perlc.c.afterlday. 

Length  of 

espoaurv  to 

heat. 

Not 
healed. 

Heated. 

1«6 
April   7 

8  houra... 
do 

17881 

G.  P.  4092 

4.020 
5,880 
2.H,'iO 
1.000 
4.  MO 

as.  000 

2,560 
2.860 
1.440 
90.400 
1&,120 
1.740 
8.880 

7S.800 
30.800 

tuo 

1.180 

16.500 

« 

400 

280 

320 

» 

800 

1.280 

• 

49.000 

36.400 

96,600 

l.lflO 

a  OB 

X 

440 
4.410 
4,200 

11.200 

1.840 

28.600 

10  minutep. 

do 

B.  roli 

Do. 

June  12 

i  hour 

4  dajrs 

1742X1 

do 

F.ae 

80  minutea. 

do 

Do. 

do 

Do. 

10  days... 
t  hour .... 

...  .do 

do :.. 

Do. 

do 

Crawford 

Do. 

do 

do 

Do. 

Do. 

10  davB . . . 

do 

do...  

O.  !•.  4OT8 

Do. 

1  liuu'r 

7  day*.... 
10  day*.. . 

do 

do 

Do. 

do...  

do 

Do. 
Do. 

aThla  character  Klgnifleii  that  there  were  too  many  rolonien  to  count. 

It  would  appear  from  the  results  recorded  in  Table  XII  that  heat- 
ing for  ten  minutes  at  61'^  C.  produced  no  effect  in  the  two  te«ts  which 
were  made  in  this  manner,  one  on  the  (».  P.  469*2  strain  of  B.  chol<n'3s 
sufM^  and  the  other  on  the  colon  culture.  The  same  tenijiorature  for 
thirty  minutes  i-obbed  the  serum  of  much  or  all  of  its  power  in  8  of 
the  11  tests. 


EFFECTS    OF    HEAT    UPON    BACTERICIDAIa.PQWER. 


25 


Table  XIII. — Effects  of  heal  upon  the  bacteriolytic  power  of  normal  hog  serum, 
dravm  from  the  tail.     Temperature  employed,  62°  C. 


Blood 


Time 
after 
draw- 
ing. 

-0- 

No.  of 
hog  and 
drawing 
of  blood. 

Culture 
used  to  test 
bacterio- 
lysis. 

Num- 
ber of 

bac- 
teria 
intro- 
duced 

per 
1  c.  c. 

Number  of  bacteria  per  1  c.  c. 
after  1  day. 

Date. 

Arterial  blood,  j   Venous  blood. 

Length  of 
exposure 
to  beat. 

Not 
heated. 

H-ted.,f,?^. 

Heated. 

1905. 
June  28 

Iday ... 
2  days . . 

1733  X.... 
do.... 

F.  26 

do 

3,880 
4,920 

380 

940 
1,480 

420 
2,440 
9,880 

300 
5,420 
6,520 
2,160 
2,720 

60 

16,320 

2,200 

40 

200 

140 

9,760 

8,160 

100 

10,480 

20,160 

2,620 

2,960 

1,540 

15,200 

1,720 

3,960 

460 

300 

1,920 

9,840 

100 

320 

600 

400 

80 

5,920 

6,560 

140 

10,720 
3,360 

30  minutes. 
Do. 

do  . . . 

do 

Crawford... 
do 

a  ac    1            Do. 

1  day 

do.... 

25,080  i           Do. 

do ... . 

00 

3,080 
10,000 

Do. 

9  days 

...do 

....do 

Do. 

1  day  . . . 

2  davs . . 

do.... 

do.... 

G.  P.  4692... 
do 

Do. 
Do. 

do 

do 

July   12 

1  day  . . . 

2  days . . 

1733  XI . . . 
do.... 

F.26 

do 

220 
27.  f.80 

5,680 

41,720 

2,240 

(?) 

Do. 

14,880 

Do. 

1  day  . . . 

2  days . '. 

do.... 

do 

G.  P.  4692... 
do 

2.440  :     3,300 
2,180  !     3.340 

Do. 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 

It  will  be  seen  from  the  above  table  that  the  eflfect  of  an  exposure 
of  thirty  minutes  showed  a  marked  influence  in  some  cases  while  in 
others  it  was  apparentl}'^  without  effect.  In  these  experiments  the 
blood  was  drawn  at  the  same  operation  from  the  carotid  artery  and 
from  the  jugular  vein,  and  it  would  appear  that  the  serum  from  the 
venous  blood  was  somewhat  more  sensitive  to  the  action  of  the  heat 
than  the  serum  from  the  arterial  blood,  if  any  conclusions  are  per- 
missible from  the  somewhat  limited  number  of  observations.  In  the 
arterial  there  was  in  only  one  case  a  marked  difference  between  the 
potency  of  the  heated  and  of  the  unheated  serum  out  of  the  12  cases 
in  which  it  was  tried.  In  the  serum  from  the  venous  blood,  on  the 
other  hand,  there  was  a  marked  difference  between  the  heated  and  the 
unheated  serum  in  the  11  tc'^ts  which  were  made.  On  the  whole  the 
effect  of  heating  at  52°  C.  would  appear  to  be  uncertain;  sometimes 
heating  seems  to  have  a  marked  effect  upon  the  bactericidal  power  of 
the  serum;  at  others  it  seems  to  lack  this  effect. 


19500— No.  95—07- 


26 


BACTKRIOLYTir    POWER    OF    BLOOD   8EBUM    OF    H008. 


Tahlk  XIV. — Efffctn  iif  hfat  h;wii  thf  liarirriitlytir  jtovrr  of  lUfrmal  hog  afruui. 
draum/rom  the  tirU.     'fempmtture  emplttyed,  6S^  C 


lUood 


Date. 


M«r 


190A. 
Apr.  19 
19 
22 
22 
2S 
'» 
S 
8 
6 
6 
6 
6 
8 
8 
9 
10 
10 
11 
12 
U 
8 
8 
• 
9 
10 
10 
11 
12 
8 
8 
10 
10 
12 
12 
II 
13 
12 
13 
12 
IS 
12 
13 
12 
IS 
12 
IS 
12 
13 


Tine  after 
drawing. 


No.  o(  )k)k  mill 

(IrawiiiK  <)( 

blitud. 


July 


CiiHurf 

llMttl  to  Utit 

b«ct«rio- 
tjrala. 


Length  of  cx- 

poMire  to 

beat 


Idar I17»V O.  P.  4092. 

no ' do B.  eoll 

2iUv» I  1733  VI Q.  P.  4092. 

do I...   .do '  Jt.eoli 

Iday i  173S  VII «.  P.  4fi92. 

do ' do li.eoli.... 

173J  Vlil >  O.  r.  4«92. 

do I  B.eoH.... 

17S3IX :  O.  P.M92. 


3  houni 

do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

.....do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

do 

.....do , 

do 

do 


do 

17401 

do.... 

1740 II.... 
do.... 

1740111... 

17401V... 
do.... 

1740  V.... 

1740  VI... 
do.... 

1741  I 

do.... 

174111.... 
do.... 

1741  III G. 

do B. 

1741IV O. 

1741V B 

17421 J 


B.eoli. 
0.  I'.  4692. 
B.etili.... 
O.  I'.  4r«2. 
B.CHli.... 
O.  P.  4({92. 
O.  P.  4692. 

B.  mli 

G.  P.  4M2 . 
X,  P.  4692. 
B.nH.... 
G.  0.4602. 
i.  oli.... 
U.     .4692. 

B.      «/!.... 

4692. 


.do. 
1742  II . . 

do.. 

r42III. 

.do. 


U. 
4692. 
i.... 
1092. 


do '  1742  I  v.. 

do 1742  V... 

Iday I7S3XI^. 

2dayK do.'.. 

1  day do... 

2days do... 

1  day do... 

2  davs do . . . 

Iday 1733XI*-. 

2dnyH do... 

1  day do... 

2  days do . . . 

1  day i do... 

2  days I do . . . 


t.r, 


P.    »2. 

,«>/.  .... 

P.    J92. 


C 

B.:^i.... 
O.  P  4692. 
B.eoli.... 

F.26 

do.... 

Crawford . 

do.... 

O.  P.  4092. 

do.... 

F.26 

do.... 

Crawford . 

do.... 

O.  P.  4692. 
do.... 


oThlN  )-hnraitcr  >i^niflc«i  that  there  were  too  many  colonlea  to  coant. 
bArtetiul  blood  svrum. 
•*  Venous  blood  scrum. 


.\s  shown  hv  the  ic.sult.s  rofoidrd  in  Table  XIV,  hratinjr  at  53  C\ 
had  the  effect  in  iiio.st  ea.se.s  of  jifieutly  diinini.shin^  the  iMictericidal 
power  of  the  iicruni,  though  thi.s  i.s  not  to  lie  noted  in  all  ea.se.s,  even 
where  the  serum  was  exposed  for  thirty  minutes. 


EFFECTS    OF    HEAT    UPON    BACTERICIDAL    POWER. 


27 


Table  XV. — Effects  of  heat  upon  the  bacteriolytic  power  of  normal  hog  serum, 
drawn  from  the  tail.     Temperature  employed,  54°  C. 


Blood 


Date. 


May  26 

June    2 

May   26 

June    2 

May   29 

June    3 

May   29 

June    3 

May  29 

June    3 

5 

6 

7 

8 

9 

19 

5 

6 

7 

8 

9 

19 

5 

6 

7 


19 
May  23 
23 
23 
24 
24 
24 


Time  after 
drawing. 


3  hours  , 


No.  of  hog  and 

drawing  of 

blood. 


1742  VIII 


....do.. 
....do.. 
...  do.. 
1742  IX. 


7  days  . 
3  hours 
7  days . 
3  hours 

5  days \ do 

3  hours do 

5  days do 

3  hours ' do 

f)  days do 


4  hour 1742  X.. 

1  day do  .. 

2  days do  .. 

3  days do  .. 

4  days do  ..- 

14  days do  .. 

4  hour ...,.  .do  .. 

1  day do  .. 


2  days . . 

3  days . . 

4  days . . 
14  days  , 
i  hour . . 


..do. 
..do. 
..do. 
..do. 
..do. 


1  day j do  .. 

2  days \ do  . . 

3  days j do  .. 

4  days do  .. 

14  days  ..■ do  .. 

do I  1742  VI  . 

do I do  .. 

do do  .. 

do I  1742  VII 

do ! do  .. 

do I do  .. 


Culture  used 

to  test 
bacteriolysis. 


Crawford . 
do.... 

F.  26 

do.... 

do.... 

do.... 

Crawford . 
do.... 

G.  P.  4692. 
do.... 

F.  26 

do.... 

do.... 

do.... 

do.... 

do.... 

Crawford . 

do.... 

do.... 

do.... 

do.... 

do.... 

G.  P.  4692. 

do.... 

do.... 

do.... 

do.... 

do.... 

do.... 

Crawford . 

F.  26 

G.  P.  4692. 
Crawford . 
F.  26 


Number  of 
bacteria  in- 
troduced 
perl  c. c. 


320 
1,9<;0 
80 
3,280 
1,740 
1,220 
2, 320 
1.060 
1,920 

700 
2,840 
2,240 
3,440 
2,200 
1.360 
4,040 
8,380 
5,200 
7,440 
3,500 
2,640 
1,440 
1,380 
3,040 

180 

360 
1,H80 
1.740 

10,  aso 

40, 500 

19,040 

1.682 

1,808 

12,040 


Number  of  bacteria 
per  1  c.  c.  after  1  day 


Not 
heated. 


Heated. 


0 
380  1 
1,960 
2,980  I 
4,480  I 
1,180  , 
0 
0 

960 : 

1,100 

26,640 

51,760 

900 

1,340 

18, 280 

a  30 

180 

120 

760 

3,160 

1,380 

00 

4,420 
0,320 
3.420 
2,000 


1,160 
2.380 
5,280 
9,360 

13,600 
1,180 
9, 520 
1,880 
5,040 
1,260 
119, 700 

41,840 
6,440 
7,280 

20,480 

00 

28,000 

8,3-20 

13,000 

48,000 


4,820 
27,760 

CO 

17,960 


Length  of 

exposure  to 

heat. 


3.376 

13,180 

2,640 

58,800 

18,280 

00 

0 

2,720 

180 

7,600 

1,640 

26,320 

10  minutes. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 

Do. 
30  minutes. 

Do. 

Do. 

Do. 

Do. 

Do. 


a  This  character  signifies  that  there  were  too  many  colonies  to  count. 

As  i.s  evident  from  the  preceding  table  the  effect  of  expoisure  for  ten 
minutes  at  54°  C,  as  well  as  exposure  for  thirt}^  minutes  at  the  same 
temperature,  is  practically  in  all  cases  to  weaken  or  suspend  the 
bacteriolytic  power  of  the  serum.  The  few  exceptions  to  be  seen  in 
the  table  are  without  significance  in  view  of  the  many  cases  in  which 
the  serum  lost  in  power. 

In  summing  up  the  results  of  heat  upon  the  bacteriolytic  power  of 
normal  hog  serum  it  would  seem  evident  that  temperatures  below  54° 
C.  are  uncertain  in  action  for  the  lengths  of  time  employed,  but  that 
54°  C.  even  for  ten  minutes  serves  quite  uniformly  to  suspend  or  at 
least  to  weaken  the  bacteriolytic  power  of  the  blood  serum.  A  num- 
ber of  tests  were  made  at  55°  C.  and  56°  C,  but  as  these  did  not  show 
any  results  that  were  not  to  be  expected  from  those  at  54°  C.  it  would 
seem  superfluous  to  give  them  in  detail. 

EFFECTS  OF  INJECTING  CUIiTXJRES  OF  B.  CHOLERA  SUIS  ITPON 
THE  BACTERIOLYTIC  POWER  OF  HOG  SERUM  FOR  THIS  OR- 
GANISM. 

Very  conflicting  results  have  been  obtained  by  various  investiga- 
tors in  regard  to  the  effects  produced  upon  the  liacterioh'tic  power  of 
blood  serum  by  the  injection  of  animals  with  cultures  of  bacteria. 
Some  have  failed  to  note  any  effect  of  such  injections,  either  to  in- 


28 


HACTERlOLVTir    I><)WKR    OF     BLOOD   HRRUM    OF    H008. 


cn>ast'  or  diiniiiish  tin*  iMU'UTiolytif  jM>ti'Hfy;  others  have  found  that 
tho  |K>tt»ncy  wrh  diminisluHl,  otliors  af^ain  that  It  wa,s  inciva-MMl.  I'n- 
(lt>r  tho  supposition  that  this  disfn'pancv  in  results  was  duo  |H'rhap8 
t«)  tho  Uvst.s  havin;,'  Im'i'u  lujuh*  at  <litf»'rcnt  int^^rvals  of  tiuH^  after  in- 
j«>clion.  and  tliat  th<>  serum  taken  from  the  same  animal  at  ditTerent 
jM-riods  mijfht  exhibit  differeni-es  in  potency,  or  that  it  uiiyfht  at  cer- 
tain tiujes  be  devoid  of  |M>wer,  tests  were  made  of  serum  tjiken  at 
various  intervals  after  one  or  more  injections.  The  results  of  these 
exjK^riments  an*  sununarized  in  the  followinj^  tjiblcs. 

Tahlk  XVI.  —  lindrriitliilir  jMurr  of  inrum  from  tht  mme  funj  (A'o.   1764)  hejort  and 
iij'ter  iuj«vtioii  with  li.  cholern  anin,  cttUure  G.  P.  4*192. 


(Iilliiri-  iwwl  to  ti<»t 
Uirleriolyni?!. 


Cnwford 

Do... 

Do... 

Do... 
F.  26 

Do... 

Do... 

Do... 
(t.  P.  4«n 

Do... 

Do... 

Do... 


Tfmv  I>Iu(mI  w«h  drawn  rvlaUvc  to  Injection. 


Before  injection 

S  dtivK  af tiT  tlrxt  injwtion 

9  diiys  Hftcr  second  inji-ction. 
lUdiiv!*  Hft<T  tliird  Injection.. 

Before  injection 

Sdiiys  after  Hrst  injection 

9  ilnys  after  !««»«>ond  injection. 

10  dayc  lifter  til ird  injection. . 

Before  iiije«'tion 

3 days  after  llrst  iniection 

9  days  after  secoiuf  injection, 
todays  after thinl  injection.. 


Number  of      Number  of 

tiNcteria  in-  I  I«cter1a  |K?r 

tn¥iH«H-<l        t  c.  c.  after 

per  1  c.  c.  1  day. 


2.180 
6,200 


16.700 
7.600 
K,200 
1.060 
6»i.400 
6,340 
4,100 


0 
0 


11.800 
2,640 

26.700 
3.800 

12,  WO 
1.020 

16.700 


The  results  given  in  Table  XVI  were  obtained  with  the  serum  from 
a  hoj^  which  was  injected  at  variou.s  intervals  .sul>cutaneousW  with  a 
lHM>f-broth  suspension  from  an  agar  culture  of  the  G.  P.  4tU)2  strain. 

The  first  injection  was  made  with  5  c.  c.  of  a  suspension  equivalent 
in  density  to  a  twenty-four-hour  typhoid  culture  diluted  about  one- 
half.  The  second  injection  was  made?  with  10  c.  c.  of  a  .suspension  of 
about  the  .same  strength.  The  third  injection  was  made  with  a  sus- 
pension consisting  of  the  entire  growth  in  twenty-four  hourtt  of  an 
agar  culture.  I^^fore  each  injection  the  bloo<l  was  drawn  from  the  tail 
of  the  animal  and  placed  on  ice  for  three  days.  After  this  the  serum 
was  drawn  off  from  the  blood  and  tested  on  the  three  .strains  in  all 
cases  except  the  .serum. from  the  blood  taken  at  the  tirst  drawing,  which 
was  tested  only  upon  the  culture  with  which  the  animal  was  sul)se- 
quently  injected — culture  G.  P.  4692. 

As  there  were  no  tests  made  with  culture  Crawford  before  the 
animal  was  injected,  it  is  impo.ssible  to  be  sure  w  bother  it  was  Imc- 
tericidal  for  this  strain  at  this  time  or  not,  but  judging  from  itsaction 
on  (i.  P.  4*)D2  iM^fore  injection  it  would  seem  prol«ble  that  it  would 
have  l)een  bactericidal  or.  at  least  inhibitory  for  culture  Crawford, 
since  the  latter  strain  was  (juite  uniformly  more  sensitive  to  the 
germicidal  action  of  .serum  in  other  experiments.  The  serum  after 
tho  inje<'tions  possessed  ipiite  .strong  liacteriolytic  properties  for  cul- 
ture Crawford. 


INJECTIONS   OF   CULTURES    OF   B.    CHOLEEiE   8UIS. 


29 


It  is  also  possible  only  to  surmise  from  the  results  with  G.  P.  4692 
that  the  serum  probably  had  at  least  inhibitory  power  for  F.  26  before 
the  animal  was  injected.  It  seems  to  hav^e  retained  its  inhibitory 
power  after  the  first  injections  for  this  organism,  but  to  have  lost  much 
in  potency  after  the  third  injection.  With  culture  G.  P.  4692  the 
results  seem  to  be  more  or  less  in  accord  with  those  obtained  with  F. 
26,  though  it  is  true  that  three  days  after  the  first  injection  the  serum 
exhibited  very  strong  bactericidal  power  for  G.  P.  4692 — stronger 
than  for  F.  26.  Still  the  results  can  not  be  compared  too  closely  in 
these  two  cases,  since  the  number  of  bacteria  introduced  differed 
widely. 

Table  XVII. — Bacteriolytic  power  of  serum  from  the  same  hog  ( No.  1755)  before  and  after 
injection  with  B.  cholerse  suis,  culture  F.  26. 


Culture  used  to  test 
bacteriolysis. 


Time  blood  was  drawn  relative  to  injection. 


Number  of 

bacteria 
introduced 
per  1  c.  c. 


Number  of 

bacteria  per 

1  c.  c.  after 

Iday. 


Crawford . 

Do.... 

Do.... 

Do.... 
F.26 

Do.... 

Do.... 

Do.... 
P.  4692 . 

Do.... 

Do.... 

Do.... 


G 


Before  Injection 

3  days  after  first  injection 

9  days  after  second  injection. 

10  days  after  third  injection. . 

Before  injection 

3  days  after  first  injection 

9  days  after  second  injection . 

10  days  after  third  injection. . 

Before  injection 

3  days  after  first  injection 

9  days  after  second  injection . 

10  days  after  third  injection. . 


2,180 
240 

5,200 

800 

16,700 

7,600 

8,200 


40 

0 

0 

47,400 

3«0 

11,880 

660 


638.400 
6,340 
4,100 


9,800 
3,800 
11,200 


The  results  of  the  experiments  with  the  serum  of  hog  1755  are 
recorded  in  Table  XVII.  In  this  experiment  the  injections  were  made 
and  the  tests  applied  precisely  as  with  hog  1754,  except  that  culture 
F.  26  was  used  for  injecting  the  animal  instead  of  culture  G.  P.  4692. 
The  concentration  and  amounts  of  culture  were  the  same  in  both  experi- 
ments, and  the  methods  of  procedure  otherwise  were  the  same.  The 
serum  from  the  hog  before  injection  was  tested  upon  the  F.  26  strain 
only;  after  injection  it  was  tested  with  all  three. 

For  the  Crawford  culture,  as  already  stated,  there  was  no  test  of  the 
serum  made  before  the  injection  of  the  animal.  The  tests  made  with 
this  organism  after  each  of  the  three  injections  showed  that  the  serum 
possessed  considerable  potenc}',  and  there  seems  no  evidence  that  the 
injections  were  followed  by  diminution  of  potency  for  this  organism. 

For  F.  26  culture  the  serum  seems  to  have  had  but  little  potency  to 
start  with  before  injection,  but  it  seems  rather  to  have  gained  than 
lost  in  potency  for  this  organism  after  injection. 

For  G.  P.  4692  culture  the  results  of  the  three  tests  are  somewhat 
diflScult  of  comparison,  perhaps,  but  the  serum  after  the  third  injection 
would  appear  to  have  been  somewhat  less  potent  than  that  after  the 
second,  and,  also,  after  the  first,  if  the  difference  in  the  initial  doses 
in  these  two  cases  may  be  disregarded  as  a  factor  in  determining  the 
results. 


30 


BACTERIOLYTIC    PoWKR   OK    BLOOD   SKRITM    OK    HOOft. 


T\HiK   WFII. — Ritderiolittir  poitrr  of  ferum  from  the  aamr  hog  {No.  1766)  before  and 
after  injection  with  h.  choterx  $ui»,  culture  Oraw/ord. 


C'uKurp  ii)w<1  to  tM( 
baeleiiolyiiix. 


Time  bluod  wm  drawn  ivlalive  to  injection. 


Cra wlord , . .  i  Before  Injwtlon 

llo..... I  3  (Ih.vx  iidcr  tinit  iiiji^-lion 

iHi > 9  <l«  VH  H I ItT  MToiul  iiije<-t inn . 

riu I  10c1av.<iH(t(<r  thini  injection.. 

K.  JB :  Ik'fon-  injtH-tion 

Ik> i  S<lH>!<HfU>r  flml  InJiH'tlon  .... 

Do \  9dny<iiirtfr  MH-oriil  iiiji'ollon. 

IHt '  10<lHVwnrU'r  tliini  iiijtM-tion.. 

«i.  I".  IW.' I  Bt'fon-  Injo'tiiin 

|l<>... I  Sdnyx  niter  (Irxt  inifrtion  .... 

|io 9(1ii>'Hafii-r  ^M-oii(|  itijirtion. 

Do 10(Uyaa(u>rttiird  injection.. 


Nuni>H>ro(      NuuiImtoI 
IwrleriH       lMcU>ri«  per 
intfoduced  I  I  c.  v.  milvr 
perle.c.    i       1  day. 


TOO 
40U 
210 


0 

0 
SIM) 


K.700 
7,600 
8.200 


«3H.400 
6, 340 
4.100 


i,»0 
2A.000 
'£2.700 


5,»eo 

0 

8,aoo 


III  tlio  oxi)»M*im«»nt  with  tlio  .siTuin  of  the  hl(KKi  of  hop  17r)(>,  the 
result-s  of  which  arc  jjivcn  in  Table  XVIII,  the  method  of  piocodme 
was  the  .same  as  with  the  serum  of  ho^  1754  and  1755,  ju.st  de.scrihe<l, 
the  only  dilTerenee  lM»ing  in  the  .strain  of  orj^nisni  employed.  In  the 
ease  of  hog  175G  the  Crawford  culture  wa.s  u.sed,  and  the  .serum  was 
tested  upon  this  organism  only  Ix^fore  injection  and  on  all  three 
strains  after  the  injections. 

With  the  Crawford  culture  the  .serum  seems  to  have  been  unaf- 
fected by  the  injections  of  the  bog.  It  seems,  at  any  mte,  not  to  have 
lost  in  potency,  and  from  the  result  after  the  second  injection,  if  any 
conclusion  is  justitiable,  it  seems  possiblj'  to  have  gained. 

With  F.  26  itvseems  to  have  lost  in  potency,  if  the  result  obtained 
after  the  first  injection  is  compared  with  tho.se  after  the  other  two. 

With  G.  1*.  4t>l>2  culture  the  serum  seems  to  have  lost  in  potency 
after  the  third  injection,  but  it  is  uncertain  whether  it  gained  or  lost 
after  the  first  two. 

Tabi.k  XIX. — liticterioliftlc  poirer  of  ncrtim  from  the  mine  hog  (iVo.  179S)  before  and 
after  injection  with  li.  rholern-  tniiti,  culture  F.  26. 


Culture  uaed  to  teat 
iMurterlolyai*. 


KM... 
Do. 
Do. 
Do. 


Time  blood  wm  drawn  relative  to  Injection. 


Before  inie<;tion '. . 

10  dayH  alter  tlrat  inkftlon  . . . 
9  dayii  after  Hecoml  injection . 
14  days  after  third  injection.. 


Nutnbi-r  of  i   Number  of 
bacteria       bacteria  pi-r 
introduced     1  c.  c.  after 
per  1  c.  c.    I        1  day. 


2.«» 
8.400 

&,aao 

2.400 


1.900 

7.-iOO 

S8.000 

W.600 


In  the  e.x|>eriment  with  the  serum  of  hog  1798,  Table  XIX,  the 
animal  was  given  three  intravenous  injections  of  suspensions  in  Ijeef 
broth  from  Jigar  cultures  of  the  F.  26  strain.  The  tirst  one  was  made 
with  a  very  dilute  suspension,  weaker  than  would  correspond  with  a 
twenty-four  hour  beef-broth  typhoid  culture,  and  the  other  two  injec- 
tions were  made  with  similar. suspensions. 

It  would  appear  from  the  re.sults  given  in  the  table  that  the  serum 
wa-s  not  actively  bactericidal  before  the  injection  of  the  animal,  but 
it  seems  to  have  had  some  inhibitory  power.     Ten  days  after  the  tirst 


INJECTIONS    OF    CULTURES    OF    B.     CHOLER^E    BUIS. 


31 


injection  there  seems  to  have  been  no  striking^  change.  The  serum 
was  still  inhibitory,  but  was  neither  markedlj'  bactericidal  nor  can  it 
be  said  to  have  been  devoid  of  potency.  But  nine  days  after  the 
second  injection,  and  fourteen  days  after  the  third  injection,  the  serum 
seems  to  have  lost  greatl}'  in  the  inhibitory  power  which  it  possessed. 
It  would  appear  that  in  this  case  the  injections  had  the  effect  of  ulti- 
mately diminishing  the  power  of  the  serum. 

Table  XX. — Bacteriolytic  power  of  serum  from  the  same  hog  (No.  1809)  before  and  after 
injection  with  B.  cholerse  suis,  culture  G.  P.  4692. 


F.  26 Before  injection 

Do !  14  days  after  injection 

Crawford I  Before  injection 

Do :  14  davs  after  injection 

G.  P.  4692 Before  injection 

Do 14  days  after  Injection 


5,920 
7,300 
5,700 
13,000 
1,140 
7,300 


5,900 
1,800 
0 
66,600 
10,9C0 
7,900 


In  the  experiment  giv^en  in  Table  XX  the  serum  of  hog  1809  was 
tested  on  the  three  different  strains  of  B,  cholerse  suis  before  the  animal 
was  injected  and  on  the  same  cultures  fourteen  days  after  the  hog  had 
received  subcutaneously  about  5  c.  c.  of  a  dilute  suspension  from  an 
agar  culture  of  culture  G.  P.  4692.  In  the  tests  made  with  the  serum 
upon  the  F.  26  strain  there  seems  to  have  been  a  slight  diminution  of 
potency  after  the  inoculation,  but  the  difference  in  the  numbers  of 
bacteria  surviving  bacteriolysis  in  the  serum  from  the  hog  before 
injection  and  those  surviving  after  injection  is  not  large  enough  to 
permit  of  very  definite  conclusions.  The  same  may  be  said  in  regard 
to  the  organism  with  which  the  hog  was  injected — culture  G.  P.  4692 — 
but  the  results  in  this  case  are  perhaps  somewhat  more  significant. 
It  would  seem  justifiable,  however,  to  conclude  that  there  was  no 
marked  increase  nor  decrease  of  bacteriolytic  power  for  F.  26  culture 
or  for  G.  P.  4692  produced  by  the  injection.  The  result  with  Craw- 
ford seems  to  show  plainly  that  the  serum  was  less  potent  for  this 
organism  after  injection  than  it  was  before. 

Table  XXI. — Bacteriolytic  power  of  serum  from  the  same  hog  (No.  18S6)  before  and 
after  injection  uith  B.  cholene  suis,  culture  F.  26. 


Culture  used  to  test 
bacteriolysis. 


Time  blood  was  drawn  relative  to  injection. 


Crawford |  Before  injection 

Do 1  hour  after  injection 

Do 3  days  after  injection . 

F.  26 Before  injection 

Do 1  hour  after  injection 

Do j  3  days  after  injection. 

G.  P.  4692 Before  injection 

Do I  1  hour  after  injection 

Do I  3  days  after  Injection . 


Number  of 

Number  of 

bacteria 

bacteria  per 

introduced 

1  c.  c.  after 

per  1  c.  c. 

1  day. 

32,200 

a  3, 840 

32,200 

a5,340 

1,560 

b  at 

30.400 

a  247. 800 

30,400 

boc 

5,000 

OC 

&5,000 

a  47.4C0 

35,000 

a  36, 600 

6.800 

<» 

a  Serum  diluted  in  the  proportion  of  1.4. 

b  This  character  signifies  that  there  were  too  many  colonies  to  count. 


32 


BACTERIOLYTIC   POWER   OF    BLOOD   SERDM    OP    HO(J8. 


In  tho  ex])oriincnt  with  tho  Hcntm  from  the  blood  of  hog  1H^)G,  given 
in  Table  XXI,  the  unimal  wjis  inj^'cU'd  iiitrftvenously  with  a  siupen- 
sion  from  an  agar  culturi'  of  .stniin  F.  20  of  about  tho  density  of  a 
34-hour  l>oof-bri>tli  culture  of  the  typhoid  bacillus.  Itefore  inje<'tion 
the  hog  was  bled  to  obtain  serum  for  testing  the  power  of  the 
nonnal  serum.  The  amount  of  blood  obtained  l)eforo  injection  and 
that  obtuine<l  one  hour  after  injection  wjis  insufficient  for  the  test  to 
be  made  with  the  quantity  of  serum  usually  employed,  so  the  senim 
was  diluted  in  l)oth  of  these  cases  with  three  parts  of  physiological 
salt  solution  to  one  part  of  serum.  There  were  no  tests  made  with 
the  undiluted  s<»rum. 

With  the  Cniwford  culture  the  serum  seems  to  have  been  quite 
potent  before  injection,  and  also  one  hour  after  injeirtion,  but  three 
days  after  injection  it  appears  to  have  been  entirely  devoid  of  bacter- 
iolytic power  for  this  organism. 

With  F.  2<l — the  strain  used  to  inject  the  animal — the  serum  had 
onl}'  weak  potency  before  injection,  and  if  there  was  any  effect  pro- 
duced b}'  the  injection  it  was  in  the  direction  of  weakening  tho  potency 
of  the  serum  for  this  organism.  This  apparent  diminution  of  potency 
is  to  be  noted  even  in  one  hour  after  the  injection,  and  is  still  more 
pronounced  after  three  days. 

With  the  G.  P.  4092  strain  the  serum  was  apparently  inhibitory  at 
the  start  befoi-e  injection  and  one  hour  afterwards,  but  three  days 
after  injection  it  seems  to  have  lost  even  the  power  to  inhibit  the 
growth  of  the  organism. 

The  results  of  this  experiment  seem  to  show  that  in  this  case*  the 
effect  of  injecting  the  hog  was  to  suspend  the  bactericidal  power  of 
the  serum  for  the  homologous"  organism  as  well  as  for  the  other 
strains  upon  which  it  was  tested. 

Table  XXII. — Bacteriolytic  power  of  serum  from  Uie  mme  hog  (Xo.  1839)  before  and 
after  injection  trith  B.  choleric  guis,  culture  G.  P.  469f. 


Culture  used  to  test 
twcteriolysis. 


Time  blood  was  drawn  relatlTe  to  Injection. 


Crawford Before  Inioction 

Do I  2  huuni  after  Injection 

Do 1  day  alter  injection  .. 

Do 1  2  day<4  alter  Injection  . 

F.  26 B«-fore  iiiiectloii 

Do {  2hoiirHaiier  injection 

Do ;  1  day  after  inje<-tiun  .. 

Do 2dav«  after  injection.. 

O.  P.  4492 Hclore  inieetlon 

Do I  2  hours  niter  injection 

Dof 1  day  alter  in je<-tlon  .. 

Do 2  days  alter  injccuon  . 


Number  of 

bacteria  in- 

ttoduced 

per  1  c.  c. 


8.760 

8.760 

600 

600 

1,020 

1,020 

840 

SM 

2.  MO 

2,840 

200 

200 


Number  of 

bacteria  {H-r 

1  c.  c.  after 

Iday. 


80 

1.400 

266,400 

18.000 

2,900 

80 

39,000 

86.400 

8.800 

9,200 

64,600 

12.400 


a  HomoUifciuu  It  the  term  now  nnivenwlly  employed  to  denote  tbe  organism  with  which  the 
animal  in  any  iciven  ease  is  injected,  as  in  the  present  case. 


INJECTIONS    OF    CULTURES    OF    B.    CHOLERiE    SUIR. 


33 


In  the  experiment  given  in  Table  XXII  the  animal — hog  1859 — was 
injected  intravenously  with  5  c.  c.  of  a  suspension  of  culture  G.  P. 
4692.  The  suspension  was  made  in  beef  broth  from  a  24-hour  agar 
culture  as  usual,  and  was  equivalent  in  density  to  a  24-hour  beef-broth 
culture  of  the  typhoid  culture  diluted  with  about  equal  amounts  of 
broth.  Tests  were  made  of  the  potency  of  the  serum  from  the  blood 
of  the  animal  before  injection  and  after  injection  with  all  three  strains 
of  B.  cholersR  suis.  Owing  to  the  fact  that  the  animal  was  killed  in  a 
moribund  condition  two  days  after  injection,  it  was  impossible  to  make 
tests  with  the  serum  from  the  blood  later  than  the  second  day  after 
injection. 

It  would  seem  hardly  necessary  to  discuss  the  results  with  the  differ- 
ent organisms  in  detail,  since  they  all  appear  to  point  to  a  lessening 
of  the  potency  one  and  two  days  after  injection.  Before  injection  the 
serum  from  the  blood  seems  to  have  been  bactericidal  for  the  Crawford 
strain  onl}-;  still  for  the  other  strains  it  was  at  least  inhibitory,  whereas 
after  injection  it  had  lost  much,  if  not  all,  even  of  its  inhibitor}^  power 
on  the  first  and  second  day  after  injection.  In  the  tests  made  two 
hours  after  injection  the  results  appear  somewhat  discrepant;  the 
serum  seems  to  have  lost  potency  for  Crawford,  and  gained  for  F.  26; 
but,  after  all,  the  figures  are  perhaps  not  very  significant  in  these 
cases. 

The  results  in  this  experiment  may  have  been  complicated  by  the 
fact  that  the  animal  from  which  the  blood  was  drawn  was  suffering 
from  acute  infection  with  B.  choleras  suis  at  the  time  the  blood  was 
obtained.  It  may  not  be  correct  to  regard  this  serum  as  immune  in 
the  sense  in  which  this  term  is  usually  understood — that  is  to  say, 
serum  from  the  blood  of  an  animal  given  injection  of  bactei  'a  and  re- 
covered. Nevertheless,  the  results  are  perhaps  not  without  interest, 
since  they  show  certainly  a  very  marked  difference  between  the  serum 
from  the  blood  of  the  hog  before  injection  and  that  from  the  blood 
after  injection. 


Table  XXIII. — Bacteriolytic  power  of  serum  from  the  same  hog  {No.  1860)  before  and 
after  injection  with  B.  choJerse  suis,  culture  F.  26. 


Culture  used  to  test 
bacteriolysis. 


Crawford . 

Do.... 

Do.... 

Do.... 

Do.... 
F.26 

Do.... 

Do.... 

Do.... 

Do.... 
P.  4692 . 

Do.... 

Do.... 

Do.... 

Do.... 


G 


Time  blood  was  drawn  relative  to  injection. 


Before  injection 

1  day  after  injection. . 

2  days  after  injection. 

3  days  after  injection. 

4  days  after  injection. 

Before  injection 

1  day  after  injection.. 
"2  days  after  injection. 

3  days  after  injection . 

4  days  after  injection. 
Before  injection 

1  day  after  injection.. 

2  days  after  injection. 

3  days  after  injection . 

4  days  after  injection. 


Number  of 

Number  of 

bacteria  in- 

bacteria per 

troduced  per 

1  c.  0.  after  1 

1  c.  c. 

day. 

1,460 

0 

1,240 

20 

140 

aoe 

5,400 

00 

•      1,620 

'       0 

2,100 

80 

700 

880 

1,100 

X 

1.500 

00 

2,260 

5,580 

10,200 

17,400 

4,100 

60 

1,500 

00 

9,900 

00 

1,380 

1,020 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 


34  HACTKRIOLYTIC    POWER    OF    BLOOD   ^KKt  M    t)K    HiKiB. 

In  tl)oox|M'riiiHMit  ivcoitU'd  in  Tul>l«'  XX II  I,  tlH>  iiniinal  lioj^  1K60 — 
wuM  iiijccttHl  tiiNt  witli  iilKJiit  2  c.  c.  of  a  (lilut<'  susiM'iiMion  of  an  a^r 
culture  in  bcof 'broth,  hut  must  of  the  nmtcrial  was  injected  merely 
under  the  skin,  as  the  attempt  Jo  strikt'  a  vein  was  not  successful. 
Two  days  after  the  tirst  injection  a  second  injection  of  atxiut  *2.5  c.  c. 
of  a  similar  suspension  from  the  same  stniiii  of  l>acterium,  in  this  case 
all  of  the  susjxMision,  was  introduced  into  tlu-  vein.  Two  days  after 
the  second  injection  a  third  inji>(*tion  was  made  similar  in  all  respects 
to  the  second.  The  suspensions  for  all  three  of  the  injections  were 
heated  at  r>7"  to  58^  C.  for  thirty  minutes.  The  blood  was  drawn  from 
the  animal  before  injection  and  on  the  lirst,  second,  third,  and  fourth 
days  after  the  last  injection.  The  result*  seem  to  Imj  (juite  uniform  for 
the  serum  on  the  second  and  third  days  after  the  inj«'ctions:  on  Inith 
days  the  serum  showed  apjMirently  entire  loss  of  bactericidal  p'»wer  for 
all  three  orj^nisms.  On  the  fourth  day  after  the  last  injection  the 
serum  had  apparently  j^ained  in  potency,  particularly  for  the  C'mwfonl 
stniin.  The  results  obtained  with  the  serum  one  day  after  the  last 
injection  are  not  very  striking,  but  the^-  perhaps  j>erniit  of  the  conclu- 
sion that  the  serum  had  not  last  in  potency  at  least  ta  any  marked 
extent. 

As  previously  stated,  the  results  of  the  injection  of  animals  with 
cultures  or  with  the  produ<'ts  of  jjrrowth  of  bacU'ria  have  lx»en  most 
contlicting  in  various  hands  in  so  far  a.s  the  effect  of  such  injections 
upon  the  bacteriolytic  properties  of  the  blood  serum  from  the  animal 
in  test-tul>e  experiments  is  <'oncerned.' 

A  numl)er  of  investigators  have  found  that  the  injection  of  an  ani- 
mal with  cultures  or  with  the  products  of  growth  of  certain  Inicteria 
increases  the  bactericidal  jK^wer  of  the  blood  serum  for  the  homologous 
organism.  Others  have  found  that  there  is  no  effect  upon  the  Imcteri- 
cidal  potency  of  the  serum  produced  by  such  injections,  while  still 
othei-s  have  found  diminution  or  complete  loss  of  power  for  the 
homologous  organism  to  follow  the  injections.  Among  tJthers  Neisi*er 
and  Wechsl)erg'*  have  found  that  the  so-calle<l  inunune  serum — i.  e., 
the  serum  froni  an  animal  which  has  received  injections  of  certain 
liacteria — no  longer  jwssesses  bjictericidal  |X)wer  for  the  homologous 
organisu)  when  the  serum  is  undiluted.  The  residts  with  the  dilutcnl 
serum  will  be  discussed  later.  Huxtt)n*  has  found  that  the  serum 
from  the  blood  of  a  normal  rabbit  is  caiMible  of  destroying  very  large 
numbers  of  the  typhoid  and  panityphoid  bacilli,  but' that  after  the 
rabbit  has  been  injecttni  with  these  organisms  it  no  longer  kills  any  of 
the  homologous  strain. 

Rut  while  there  is  undoubtedly  conflict  in  regard  to  the  matter,  the 
preponderance  of  evidence  certainly  seems  to  show  that  the  serum  is 
frequently,  if  not  always,  w«»akened  in  Iwctericidal  |X)wer  or  com- 
pletely robljed  of  this  power  for  the  homologous  organism  by  these 


EFFECTS    OF.  DILUTION    UPON    BACTERIOLYTIC    POWER.  35 

injections,  and  the  results  recorded  above  seem  in  many  instances  to 
bear  out  this  result  in  the  case  of  hogs  injected  with  B.  clwleriB  suls. 
But  it  would  also  seem  apparent  that  there  is  a  tendency  in  some  cases 
to  a  restoration  of  the  lost  potency  on  further  injection  of  the  animal. 

These  conclusions  seem  justifiable  at  least  from  the  experiments  with 
the  F.  26  and  the  G.  P.  4692  strains.  The  results  with  the  Crawford 
strain  diflfer  somewhat  in  this  respect  from  those  with  the  other  two 
strains,  and  this  may  be  due  to  the  fact,  alread}^  mentioned,  that  this 
organism  is  of  only  feeble  pathogenic  potenc}' .  It  seemed  to  be  much 
more  easily  killed  by  the  serum  generally  than  the  other  two  strains, 
and  it  probably  also  has  less  power  than  these  to  cause  the  reaction 
in  the  serum  which  is  necessary  for  the  production  of  immune  serum 
as  this  is  understood.  The  serum  of  hogs  injected  with  this  strain 
would  consequently  perhaps  be  expected  to  behave  more  like  the 
serum  from  an  uninoculated  animal.  In  some  cases  the  serum  of  the 
blood  of  hogs  injected  with  the  F.  26  or  With  the  G.  P.  4692  strain 
seemed  to  have  retained  bactericidal  power  for  the  Crawford  strain 
after  having  lost  it  for  the  other  two. 

The  variations  which  have  been  alreadj'  alluded  to  as  occurring  in 
the  serum  of  animals  from  time  to  time,  and  which  are  due  to  causes 
as  yet  obscure,  must  be  borne  in  mind  in  drawing  conclusions  from 
the  results  of  experiments  with  the  serum  from  animals  injected  with 
cultures.  Of  course  it  is  impossible  to  determine  in  the  present  case 
the  extent  to  which  this  spontaneous  variation,  as  it  is  at  present  con- 
venient to  call  it,  may  have  aifected  the  results. 

In  view  of  the  many  conflicting  results  with  the  serum  of  the  blood 
both  of  normal  and  of  injected  animals,  in  so  far  as  the  bactericidal 
properties  are  concerned^  it  is  evident  that  there  are  some  obscure  fac- 
tors in  the  process  which  the  methods  at  present  in  use  are  incapa- 
ble of  determining.  If  the  blood  serum  from  one  and  the  same  animal 
is  found  to  be  bactericidal  at  one  time  and  not  at  another,  where  the 
animal  is  kept  under  apparently  the  same  conditions,  and  if,  further- 
more, the  injection  of  cultures  is  found  by  certain  observers  to  have 
no  effect  upon  the  bactericidal  potency  of  the  serum,  by  others  to 
increase  the  potency,  and  by  others  again  to  lessen  the  potenc}^  there 
must  be  some  ver}^  subtle  factors  which  enter  into  the  problem,  else 
they  would  scarcely  be  overlooked  by  so  many  investigators. 

EFFECTS    OF   DILUTION    UPON    THE    BACTERIOLYTIC    POWER    OF 
NORMAL  AND  IMMUNE  SERUM. 

DILUTION   OF   NORMAL   SERUM. 

The  effects  of  dilution  upon  the  bactericidal  power  of  hog  serum  were 
tested  in  the  manner  usually  emplo3^ed  in  such  cases.  The  method  for 
obtaining  the  blood  was  to  cut  off  a  piece  of  the  tail  and  allow  the 
blood  to  run  into  sterile  test  tubes  or  flasks,  which  were  put  into  the 


Sfi 


BACTRRIOLYTir   POWER   OF   BLOOD   BKRUM   OF   H008. 


rcfrij^iM-ator  iiuiiiodiutoly.  Tli«'  scrum  wu.s  (imwn  off  tm  tjoon  as  it  had 
Heparatcd,  and  1  c.c.  of  it  wiu  put  into  each  of  three  tuben.  Various 
dilutions,  as  mcntionod  in  the  tahles,  were  also  prefiartnl  with  physio- 
lojjieal  .salt  .solution,  and  portions  of  these  dilutions  wer«^  distributed 
into  test  tulKi.s,  three  tulles  from  each  dilution,  each  eontaining  1  e.c. 
In  most  aises  0.1  or  0.05  e.c.  of  neutral,  jwptonized  l»eef  broth  was 
added  to  insure  suiKcient  nutrient  material  for  the  baeteria  whieh  were 
8u!)8e(|uently  intrixluced.  This  was  done  in  conformity  with  the 
method  ordinarily  employed  in  such  exiwriments.  Each  of  the  tul)es 
was  tinally  incK-ulated  with  a  carefully  measured  amount,  usually  0.05 
CO.,  of  a  susjxjnsion  in  ph3'siological  salt  solution  from  a  recent  agar 
culture.  One  series  each  was  inoculated  with  one  of  the  three  strains 
of  /?.  rhohnf  HUiK,  In  addition  to  the  tulx'S  of  undiluted  and  of  diluted 
serum,  three  tubes  of  physiological  salt  .solution  with  the  addition  of 
0.05  c.c.  of  l)eef  broth  were  also  prepared  in  each  case,  and  inoculated 
at  the  same  time  as  the  serum  tubes  with  an  amount  of  culture  equal 
to  that  u.sed  for  the  serum  tulxis.  These  tubes  constituted  the  checks. 
Immediately  after  the  inoculation  of  the  tubes,  agar  plates  were  made 
from  each  of  the  tubes  of  .salt  solution  with  carefully  measured 
amounts,  and  after  twenty-four  hours  at  room  temperature  agar  plates 
were  made  from  all  the  tubes  of  a  series — from  both  the  undiluted 
and  diluted  serum,  and  from  the  salt  .solution.  In  nearly  all  cases 
0.05  c.  c.  was  used  for  each  plate.  Tlie  colonies  were  counted  after 
twenty-four  or  forty-eight  hours  in  the  incubator  at  Ixxiy  tcMiiperature. 

Table  XXIV. — Bacteriolytic  potcer  of  normal  hog  nerum  on  dilulion. 


NaoT 

Culture  used  to  test 
bttfteriolysin. 

Number  of 

bacteria 
Introduced 
per  1  c.  c. 

Number  of  bacteria  in  aemm  after  24 
hours. 

Number 
of  bac- 
teria In 
cherkMU 
solution. 

hog. 

Undllated. 

Diluted 
lto60. 

Diluted 
ItofiOO. 

Diluted 
lto6.000. 

140 

2,280 

6,200 

2,340 

161,000 

900 

OD 

60,800 

17W 

Craw  ford 

840 
2,620 
2,660 

840 
8.400 
.   2,060 
6,700 
6,920 
1,140 

82,200 

30,400 

36,000 

8,760 

1,090 

2.840 

1,460 

2.100 

10,200 

0 
1,980 
1,680 

0 
2.040 
2,900 

0 
6.900 
10,900 

^S,S40 

^ 247, 800 

647,400 

80 

2.900 

8,800 

0 

80 

17.400 

28.400 

1.400 

2.700 

0 

36,200 

2,980 

SCO 

109. -JOO 

2,880 

81,200 

1,740 

82,800 

74,200 

aae 

1.980 

oe 

128,200 

F.26 

(J.  P.  4692 

1808 

Cn»w(ord 

F.ae 

0.  1*.  4fi92 

1809 

«• 

F.28 

» 

0.  P.  4602 

78,000 

Crawford 

Diluted 
ItoIO. 

820 

117.600 

37.200 

1.160 

13,000 

6,200 

80 

80,480 

11,400 

Diluted 
1  to  100. 

Diluted 
1  to  1.000. 

1886 

136.800 

806.000 

149.800 

187.600 

16.080 

12.500 

604,000 

61.400 

OB 

at 

OD 

ae 

CB 

118.400 
879.200 
446,200 

«■ 

F.28 

OB 

G.  I*.  4602 

OD 

1869 

Cru  w  lord 

OD 

F.26 

«• 

0.  P.  4092 

0> 

1800 

18.700 

r.M 

at 

0.  P.  4692 

206.000 

afhU  character  klgnlOes  that  there  were  too  many  colonlea  to  count. 


»DUatedl  to4. 


EFFECTS    OF    DILUTION    UPON    BACTERIOLYTIC    POWER.  37 

The  results  given  in  Table  XXIV  show  tiiat  the  serum  taken  from  a 
hog  before  injection — i.  e.,  normal  serum — as  a  rule,  loses  in  potency 
upon  dilution;  that  is  to  say,  a  given  amount  of  the  diluted  serum 
kills  actually  fewer  bacteria  than  the  same  amount  of  the  undiluted 
serum.  This  was  to  be  expected  a  priori  and  from  the  results  of 
others  with  other  organisms  and  with  other  animals.  But  the  point 
which  seems  specially  wortli}^  of  note,  and  which  will  be  discussed  in 
a  different  connection  in  this  paper,  is  that  the  diminution  of  potency 
on  dilution  is  not  in  proportion  to  the  degree  of  dilution.  Not  only  is 
this  the  case,  but  in  at  least  4  out  of  the  18  experiments  given  in 
the  table  there  was  apparently  an  actual  increase  of  power  on  dilution. 
Those  familiar  with  the  subject  will  at  once  see  the  bearing  of  these 
results  upon  the  difference  between  the  bacteriolj^tic  properties  of 
normal  serum  upon  the  one  hand  and  of  immune  serum  on  the  other. 
As  it  is  attempted  to  show  below,  the  difference  seems,  after  all,  to  be 
one  of  degree  only,  and  not  a  difference  of  kind. 

In  interpreting  the  above  results  it  must  of  course  be  borne  in  mind 
that  the  number  of  bacteria  found  in  the  serum  after  twentj^-four  hours 
probabl}'  does  not  represent  merely  the  bacteria  which  are  not  killed 
b}'  the  serum,  but  it  represents  in  addition  the  number  resulting  from 
the  multiplication  of  these  after  bacteriolysis  has  ceased.  Thus,  in  the 
case  of  the  serum  from  hog  1859,  if  1  c.  c.  killed  approximately  4,000 
of  the  Crawford  bacilli,  0.1  c.  c.  of  the  serum  should  evidently  have 
killed  400  and  have  left  3,600  to  multiply,  unless  in  addition  to  the 
bactericidal  power  the  serum  also  possessed  inhibitory  power  for  the 
bacteria  which  survived  bacteriol3'sis.  If  the  serum  had  exhausted  its 
bacteriolytic  power  in  ten  or  twelve  hours  and  exerted  no  further 
influence  after  this  time,  there  should  have  been  in  the  1  to  10  dilution 
after  twenty-four  hours  a  multiplication  of  the  3,600  bacilli  left, 
resulting  in  some  4,000,000  or  5,000,000  even  at  the  rate  of  only  one 
generation  an  hour,  as  a  simple  calculation  shows.  But  even  if  only 
one  bacillus  were  left  after  twelve  hours,  there  would  result  over 
4,000  in  twenty-four  hours,  provided  there  were  no  hindrance  to 
multiplication  at  the  rate  assumed  above.  It  would  seem,  therefore, 
that  either  relatively  more  bacilli  were  destro}  ed  by  the  diluted  serum 
than  b}'  the  undiluted,  or  that,  aside  from  the  bacteriolj'tic  action,  the 
diluted  serum  exerted  an  inhibitory  effect  in  the  experiment  quoted; 
for  at  the  end  of  twenty -four  hours  there  were  only  about  1,000  bacilli 
in  the  1  to  10  dilution  instead  of  the  4,000  calculated  as  the  miinimum 
number  possible  under  the  supposition  stated. 

The  dilution  of  1  to  1,000  in  the  same  experiment  should  evidently 
have  killed  only  40  bacilli  if  it  acted  with  the  same  relative  intensity  as 
the  undiluted,  i.  e.,  if  it  possessed  just  one  one-thousandth  of  the  potency 
of  the  undiluted  serum.  Supposing  that  all  bacteriolytic  action  ceased 
after  fourteen  hours,  the  3,960  bacilli  left  alive  increasing  at  the  rate 
of  one  generation  an  hour  would  be  represented  by  over  4,000,000 


38  BACTERIOLYTIC    1»'JWKK    OK    BLOOD   8KRIJM    OF    HOdS. 

Imc'illi  in  ten  hours,  wbcroas  as  h  umttor  of  fuel  there  were  only  uhout 
3*2,0(M>  in  this  dihition  ufU»r  t\v<Mitv-f<>ur  hours. 

The  tiuu'  at  which  l«icteriolysis  is  exhausted  in  serum  at  room  tem- 
peratures watt  not  deteiiuinetl,  but  tcsbj  made  at  the  end  of  three  or 
four  hours  showi»d  that  up  to  this  time  there  was  little  or  no  appreei- 
uhle  etToft  u|)on  the  numln'r  of  bnrteria  intrt>duced,  so  that  the  alK>ve 
assumption  that  the  process  is  exhausti>d  in  twelve  or  fourteen  hours 
may  or  may  not  ho  correct.  But  whether  it  is  or  is  not,  the  relative 
effect  of  the  various  dilutions  would  he  shown  by  assumin*^  the  proc- 
ess to  come  to  an  end  simultaneously — and  this  is  prol>ably  the  case — 
at  any  time  within  the  twenty  four  hours,  the  ol>ject  of  the  ealcula- 
tioii  beinjj  merely  to  show  that  no  definite  rule  of  pro|x>rtion  could  l>e 
observed,  and  that  the  diluted  serum  in  all  cases  destroyed  relatively 
more  bacteria  than  the  undilute<l  serum.  While  dilution  of  normal 
serum  causes  a  marked  diminution  in  the  bactericidal  power,  it  does 
not  seem  to  diminish  it  to  the  extent  that  would  l)e  ex|)ected  a  priori. 
In  other  words,  a  dilution  of  1  to  10  does  not  seem  to  destroy  just 
one-tenth  the  number  of  bacilli  that  the  undiluted  senim  destroys,  but 
a  nuich  larger  number  than  this,  or  at  least  if  it  docs  not  destroy  a 
iarjfor  nimiber  it  prevents  as  relatively  great  an  increase  as  that 
which  takes  place  in  the  undiluted  serum. 

Still  another  method  of  comparison  which  app<»ars  permissible,  and 
which  leads  to  the  sjune  conclusion,  is  to  calculate  the  number  of 
bacteria  which  should  result  in  a  dilution  of  1  to  1(X)  if  the  same  rela- 
tive increase  took  place  as  in  the  1  to  10  dilution;  or  to  compare  the 
relative  increase  in  any  of  the  dilutions  with  that  in  the  undiluted 
serum  in  the  experiment  given  above.  For  such  a  comparison  it  may 
Ih>  assumed  that  the  organisms  which  were  not  destroyed  in  fourteen 
hours  multiplied  in  the  undiluted  and  in  the  diluted  serum  at  the  same 
mte,  or  at  least  that  if  the  serum  retarded  nudtiplication  this  effect  was 
more  marked  in  the  more  concentrated  serum  than  in  the  more  dilute. 
It  would  seem  evident,  then,  that  the  relatively  smaller  number  of 
Imcteria  in  the  diluti'd  serum  can  be  accountt'd  for  only  on  the  sup- 
|x>sition  that  the  diluted  scrum  retains  more  Imcteriolytic  j)Ower  than 
would  be  ex|)ected  from  the  degree  of  potency  shown  by  the  un- 
diluted serum,  according  to  the  simple  rule  of  pro|K)rtion.  This  point 
seems  not  td"  have  been  alluded  to  in  the  literature,  but  it  would  seem 
nevertheless  not  without  significance  p«'rhaps,  since  it  tends  ap- 
parentl^to  show  that  the  difference  between  normal  serum  on  the  one 
hand  and  immune  .»<erum  on  the  other  is  merely  a  difference  of  degree 
and  not  a  difference  of  kind.  The  matter  of  the  actual  identity  in  the 
l>chavior  of  normal  sera  and  of  inmiune  sera  on  dilution,  in  spite  of 
the  apparent  difference  shown  by  them,  will  l)e  discussed  more  fully 
in  a  different  connection  below,  where  the  attempt  is  made  to  show 
that  the  law  of  complement  diversion  applies  to  normal  serum  as  well 
as  to  immune  serum. 


EFB^ECTS    OF    DILUTION    UPON    BACTERIOLYTIC    POWER.  39 

DILUTION   OF   IMMUNE   SERUM. 

The  results  obtained  by  tests  made  to  determine  tiie  effect  of 
dilution  upon  the  bacteriolytic  power  of  immune  hog  serum — that  is  to 
say,  of  serum  from  hogs  injected  with  cultures — in  the  main  agree 
with  those  observed  in  all  recent  investigations  in  this  direction 
with  the  serum  of  other  animals  injected  with  t3'phoid,  paratyphoid, 
Asiatic  cholera,  and  other  bacteria.  The  behavior  of  the  serum  under 
such  conditions  is  very  peculiar ;  it  seems  to  be  in  conflict  with  all 
laws  of  proportion  which  are  applicable  to  other  chemical  reagents, 
particularly  in  regard  to  germicidal  substances  in  general.  With  ger- 
micidal substances  in  general,  as  it  is  hardly  necessary  to  state,  the 
more  concentrated  the  germicide  the  more  powerful  the  destructive 
power.  A  strong  solution  of  carbolic  acid  is  a  more  powerful  germi- 
cide than  a  weaker  solution.  But  not  so  with  immune  blood  serum. 
Paradoxical  as  it  ma}'  seem,  the  bacteriolytic  potency  of  the  immune 
serum  increases  upon  dilution.  The  undiluted  immune  serum  may  be 
weak  or  wanting  in  bactericidal  properties,  and  yet  become  more  and 
more  potent  upon  dilution.  In  other  words,  1  c.  c.  of  undiluted 
immune  serum  may  be  found  to  kill  few,  if  any,  bacteria ;  it  ma}^  be 
found  even  to  furnish  a  good  nutrient  medium  in  which  the  bacteria 
grow  ;  whereas  1  c.  c.  of  a  dilution  consisting  of  1  part  of  the  same 
serum  to  10  or  100  or  1,000  parts  of  dilute  salt  solution  may  be  found 
to  kill  many  hundred  bacteria  introduced  into  it.  It  goes  without 
saying,  of  course,  that  there  is  a  limit  to  this  increase  of  potency  upon 
dilution.  Where  the  dilution  goes  beyond  a  certain  point  the  potency 
becomes  weakened.  There  is  for  each  immune  serum  an  optimum 
dilution  for  bacteriolysis,  above  and  below  which  there  is  less  and 
less  potency.  Thus,  as  will  be  seen  in  some  of  the  experiments 
described  below,  the  greatest  potency  in  one  case  was  in  a  dilution  of 
1  part  of  serum  to  100  parts  of  salt  solution.  In  the  case  of  a  dif- 
ferent scrum  the  optimum  dilution  for  bacteriolysis  was  1  part  of 
serum  to  1,000  parts  of  salt  solution. 

Buxton  very  appropriately  spoaks  of  the  phenomenon  just  described 
as  the  formation  of  bacteriolytic  zones,  of  which  three  may  be  recog- 
nized in  every  immune  serum — a  pro-killing  zone,  a  killing  zone,  and 
a  post-killing  zone.  The  action  of  the  undiluted  imnmne  serum  lies 
in  the  pro-killing  zone,  thatcf  the  optinmm  dilution  lies  in  the  killing 
zone;  that  of  dilutions  in  which  bacteriolysis  becomes  again  weakened 
lies  in  the  post-killing  zone.  Of  course  it  is  to  be  understood  that  these 
zones  are  not  sharply  marked  off  from  one  another,  but  that  they  grad- 
ually merge  into  each  other.  The  pro-killing  zone,  for  example,  ma}'^ 
extend  over  dilutions  of  several  strengths,  and  the  same  is  true  of  the 
killing  and  of  the  post-killing  zones.  Thus  in  some  of  the  experiments 
given  below  there  appeared  to  be  no  marked  difference  between  the 
bactericidal  power  of  the  undiluted  serum  and  the  same  serum  diluted 


40 


BACTERlOLYTir    PoWKR    oK    BLOOD   SERUM    OK    H008. 


in  the  pro|M)rtion  of  1  to  10  or  1  to  10();  the  pro-killing  zone  in  Uiese 
caseti  would  Im>  said  to  extiMid  to  the  dilutionn  of  1  to  10  or  1  to  100. 
The  zones  are  then^foro  s|K>kon  of  as  lonjfor  or  shorter,  accordin)^  to 
whether  they  apply  to  many  or  t4)  few  driiitions. 

The  |>cculiar  In^havior  of  iinnuine  Horuni  just  de8cril)ed  wa-s  tirst 
observed  by  Neisser  and  Wechslwrtf,"  and  is  called  the  Neihwer- 
VVechsljcrg  phenomenon.  While  it  is  true  that  this  phenomenon  was 
observed  quite  freipiently  in  inunune  hog  senun,  as  will  l)e  shown 
later,  it  was  not  as  notioeablr  in  some  <'ases  as  it  was  in  others,  and 
moreover  the  three  zones  varied  in  length,  not  only  after  the  injc»c- 
tion  of  different  strains  of  B.  cholerse  ttuis  in  different  hogH,  but  also 
after  the  injection  of  the  same  strain  in  different  hogs. 

It  has  already  been  shown  that  noiinal  serum  of  hogs  gains  rela- 
tively in  p>otency  on  dilution,  and,  as  has  just  l>een  stated,  immune 
serum  also  gains  in  potency.  The  two  behave  alik«',  and  at  most  there 
is  only  a  difference  of  degree  between  the  amount  of  gain  in  either 
(rase.  But  even  a  difference  of  degree  between  the  two  sorts  of  serum 
is  not  alwaj's  noticed  in  the  present  experiments,  for  the  normal  serum 
acted  in  some  cases  just  like  immune  serum.  It  was  not  only  relatively 
more  potent  on  dilution,  but  it  killed  actually  more  bacteria  per  1 
c.  c.  of  seriun  in  certain  dilutions  than  in  dilutions  containing  more  of 
the  serum. 


Table  XXV. — Efert  of  dilution  upon  immune  hoq  nrrum  a»  compared  mih  the  effeti  oj 
dilution  uptm  the  normal  serum.     Hog  1869,  mjeded  with  culture  G.  P.  46!tii. 


(rulture 

Number 

Number  of  bacteria  in  Kenim 

after  24 

Number 

used  to  tent 

Time  blood  wati  drawn 

of  bacteria 

nours. 

»)f  ba»- 
terin  in 

tNU-terii>- 

relative  to  injection. 

introduced 

lyiii*. 

per  1  c.  c. 

Cndilutn] 

Diluted                  - 

Diluted 

Diluted 

check  mil 

llolO. 

1  to  ICO. 

1  to  1,000. 

ailution. 

Cnwford . . 

Before  injection 

2  hoim  after  injertlon.. 

S,760 

80 

i.iao 

149,  WO 

a« 

OB 

S,760 

1.460 

200 

4,900 

» 

00 

1  day  af tor  injt'i-tioii 

600 

286.400 

4.600 

80 

K» 

i.fiwr 

2  davx  uftiT  injfi-tlon... 

600 

1H.000 

700 

4,eoo 

12.400 

1.580r 

F.2« 

liefon-  inletnion 

2  hours  iiitor  Injertlon.. 

1.020 

2,900 

13,000 

IK7.600 

oc 

<e 

1.020 

W 

11.400 

162,400 

OC 

«ft 

1  day  after  liij<'''tioii 

340 

8^,000 

27.  mo 

480 

6,aoo 

a, TOOT 

2  davM  after  lnje<'tion... 

840 

36,4110 

l.NOO 

9.440 

900 

2,  TOOT 

U.  F.4e92.. 

Bi'fore  init'<'tloii 

2..H40 

3,  WO 

6,-J» 

16.  OKI 

OS 

•• 

2  hoiim  iiftir  iiijertion.. 

2,840 

9,200 

27.400 

1.S.200 

«e 

» 

1  day  after  injection .... 

20O 

64,6(!0 

22,400 

19, «» 

60 

200r 

2  days  after  injci-tion. . . 

200 

12.400 

140 

400 

l.OOO 

20or 

a  Thiji  character  tdirniflea  that  there  were  too  many  colonic*  to  count. 

An  example  of  the  difference  often  to  be  seen  l>etween  the  bac- 
teri(*idal  power  of  the  serum  from  a  hog  lx»fore  and  after  injection 
with  cultures  of  B.  chohT:v  sul»^  in  .so  far  as  the  effect  of  dilution  is  con- 
cerned, is  given  in  Table  XXV.  The  animal  in  this  ca.«<e  was  injected 
with  a  culture  of  the  (J.  P.  4«U)2  .strain,  and  the  bUxKl  was  draw^n 
before  and  at  several  intervals  after  injection,  liefore  the  injection 
the  serum  from  the  blood  of  this  hog  showed  inhibitory,  but  no  bac- 
tericidal power  for  the  organism  with  which  the  animal  was  injected. 


EFFECTS    OF    DILUTION    UPON    BACTERIOLYTIC    POWER. 


41 


In  this  case  there  were  2,840  of  these  organisms  introduced  per  1  c.  c. 
into  the  undiluted  serum,  and  after  twenty-four  hours  there  were 
3,800  per  1  e.  c.  present.  In  the  dilution  of  1  to  10  the  same  number 
of  organisms  introduced  about  doubled  in  number  in  twenty-four 
hours;  in  the  dilution  of  1  to  100  the  aMiie  number  of  organisms 
increased  about  fivefold;  in  the  1  to  1,000  dilution  there  was  a  count- 
less increase.  In  the  undiluted  serum  from  the  blood  of  the  same  hog 
two  days  after  inoculation  there  was  a  sixty-two  fold  increase  of  the 
200  bacilli  per  1  c.  c.  which  were  introduced,  while  in  the  1  to  10  dilu- 
tion with  the  same  number  of  bacilli  introduced  there  were  only  140 
alive  after  twenty-four  hours,  in  the  1  to  100  dilution  only  400,  and 
in  the  1  to  1,000  there  were  1,600.  In  other  words,  the  normal  serum 
diminished  in  potency  on  dilution  while  the  immune  serum  increased 
in  potency  on  dilution.  It  should  be  noticed,  however,  that  the  dimi- 
nution of  potency  of  the  normal  serum  is  an  actual  and  not  a  relative 
decrease  on  dilution.  In  this  case,  as  in  others,  the  normal  serum 
killed  more  bacteria,  relatively,  when  it  was  diluted  than  it  did  when 
undiluted. 


Table  XXVI. — Effect  of  dilytion  upon  immune  hog  serum  as  compared  with  the  effect 
of  dilution  upon  the  normal  serum.     Hog  1798,  injected  with  culture  F.  26. 


Culture 
used  to  test 

Time  blood  was  drawn 

Number  of 

bacteria 
introduced 
per  1  c.  c. 

Number  of  bacteria  in  serum  after  24 
hours. 

Number 
of  bac- 
teria in 
check  salt 
solution. 

bacterio-        relative  to  injection, 
lysis. 

Undiluted. 

Diluted 
lto50. 

Diluted 
1  to  500. 

Diluted 
1  to  5,000. 

F.26 

Before  i  n j  ection 

10  davs  after  first  injec- 
tion. 

9  days  after  second  in- 
jection. 

14  days  after  third  in- 
jection. 

10  days  after  first  injec- 
tion. 

9  days  after  second  in- 
jection. 

14  days  after  third  in- 
jection. 

10  days  after  first  injec- 
tion. 

9  days  after  second  in- 
jection. 

14  days  after  third  in- 
jection. 

2,620 
8,400 

5,920 

2,400 

840 

5,700 

13,000 

2,660 

1,140 

7,300 

1,980 
7,200 

38,000 

36,600 

0 

160 

359,600 

1,680 

1,600 

5,100 

1,400 
106,400 

38,400 

6  86,800 

26,400 

400 

612,000 

2,700 

2,000 

69,100 

1,740 
103,600 

84,000 
6  81,200 

81,200 

27,600 
620,400 

32,800 
5,500 

6  7,500 

2,280 
6,900 

(X  00 

6112,000 
140 

00 

6175,000 

5,200 

26,600 

6oo 

Crawford . . 

00 

G.  P. 4692 

ao 

00 

73,000 

00 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 
6Dilutions  used  were  1  to  10,  1  to  100, 1  to  1,000. 

That  there  is  a  difference  in  the  behavior  of  the  blood  serum  of  dif- 
ferent hogs  injected  with  the  same  strain  of  B.  cholerse  suis  is  shown 
by  comparing  the  results  given  in  Table  XXVI,  hog  1798,  with  those 
shown  in  Table  XXVII,  hog  1860.  Both  of  the  animals  were  injected 
with  culture  F.  26.  Before  injection  the  blood  of  hog  1798  showed 
considerably  more  bacteriolytic  power  on  dilution  for  the  organism 
with  which  the  animal  was  afterwards  injected  than  the  blood  serum 
of  hogs  did  in  other  cases,  though,  as  has  been  seen,    normal  hog 


42 


HAnERloLVTIO    POWER   OK    HLOOD   HKRT'M    OF   H008. 


Henun  frtniuontly  rt^tainn  a  coiwidiM'ablo  dejjroo  of  jx)tency  u|H>n  dilu- 
tion. This  soruiii  appearH  to  have  |)osfMjHstHl  alM)iit  an  oqiial  dcj^roe  of 
pottMU'V  in  all  tho  (lihitions  whifh  wore  triod,  and  to  havo  boon  alK>ut 
a8  {)otcnt  whon  diltittHl  us  it  Wiis  when  undiluted.  Ten  duvN  after  in- 
jection it**  behavior  was  v^rv  pt'culiar,  for  it  wju-.  apimrently  alH>ut  a« 
|X)tont  as  the  nonnul  serum  when  it  was  undiluted  and  in  the  1  to 
5,000  dilution,  but  it  had  lost  pot^Micy  in  the  dilutions  of  1  to  50  and 
1  to  500.  Nine  days  afti^r  a  second  injection  it  had  apinircntly  lost  in 
bactericidal  |>otoncy,  lH)th  when  diluted  and  undiluted.  Fourteen 
days  after  a  third  injection  it  seems  to  hav«' ^'ained  in  |)ower  in  all 
strengths.  If  this  is  compared  with  the  behavior  of  the  serum  from 
the  blood  of  hog  18«>0,  Table  XXVU,  a  hog  also  injected  with  the  F. 
20  strain,  it  will  Ije  noticed  that  there  is  a  marked  ditlerence. 

Table  XXVII. — Effert  of  dilution  upon  immune  hog  »erum  a$  compared  with  the  effect  of 
diltUinn  upon  the  normal  serum.  Hog  ISHO,  iujedeil  hdrnrenotmly  on  four  auecesnve 
days  with  culture  F.  ifti,  al)out  5  c.  c.  luutedfor  thirty  minutfji  cU  58-59°  C. 


Cnltare 
uwdtotest 

Time  blofxl  wa-;  dmwn 
rwlatlvf  to  iiijwtion. 

Nnmber  of 
bneteria 

introduced 
per  1  c.  c. 

Number  oi 

Uicterla  in  Knim 
hoara. 

after  34 

Number 
of  bac- 
teria in 
cheelcMUt 
solution. 

bacterio- 
IjmU. 

UndUuted. 

Diluted 
ItolO. 

Diluted 
1  to  100. 

Dllntf^ 
1  to  1,000. 

Crawfonl . . 

Before  1  njertion 

1,460 

0 

(» 

12,500 

118,400 

18,700 

1  dnyaftiT  injei'tion 

1,-J40 

20 

14,  M» 

2,220 

6,200 

5.760 

2dHysnfttr  in  i-ction... 

140 

aoD 

» 

19,(100 

6.600 

460 

aduvsaftiT  ill  et'tioii... 

.V400 

o> 

few 

6» 

6aD 

» 

4  davHnftcr  ill  wtioii... 

1.620 

0 

0 

2.800 

14,400 

20.400 

F.a6 

Bi'fore  injei'iion 

•.',  100 

80 

80,480 

604.  iWO 

879.200 

X 

1  dayiiftor  injection 

700 

880 

2,160 

70.000 

9ft.  OUO 

10.000 

■idayxafter  inj»'<-tion... 

1,100 

90 

» 

200,200 

l.<i6.800 

64,400 

Sdavsafter  iiijiniion... 

l.-VW 

an 

b» 

froo 

6, 

OB 

Ida'vs after  injiTtion... 

2,260 

5,680 

1,820 

2,860 

6.900 

IS.  600 

O.  P.  4e»2.. 

Before  injeetioii 

10,200 

17.400 

11.400 

61,4rO 

446,200 

206,000 

1  dav  after  injeetion 

4,100 

60 

H,H00 

10,600 

98,000 

28,000 

2  davs lifter  ill  wtion... 

1,500 

x 

39 

64,400 

46,800 

4.400 

8  days  after  in  eetion... 

9,900 

« 

hy> 

b> 

6, 

QB 

4  d«y« after  injeetion... 

1,380 

'..OM 

2,100 

2.480 

10,800 

OD 

a  Thl.«  character  idfatiflm  that  there  wert>  too  many  rolonieo  to  count. 
b  Dilutions  tifMxl  were  1  to  .V),  1  to  500.  1  to  S.OtX). 

In  the  experiment  with  the  blood  serum  of  hog  1860,  Table  XXVU, 
the  animal  was  injected  on  four  successive  days  with  t|uite  dense  sus- 
pensions, considerably  denser  than  a  twenty-four-hour  culture  of 
typhoid.  The  sus|)ensions  were  heated  for  thirty  minutes  at  58-51»-  C. 
before  injection,  and  the  injections  were  made  intravenously.  On 
each  day  before  injection,  and  previous  to  any  injection,  blood  was 
drawn  from  the  t^iil  of  the  animal,  and  after  the  separation  of  the 
serum  this  was  used  on  the  three  strains  of  //.  rhoferve  suis.  Doubtless 
the  fact  that  the  tests  weW'  made  while  the  animal  was  lieing  immu- 
nized had  some  effect  u|M)n  the  results  obtained,  and  for  this  reason 
they  are  different  in  .some  niea-sure  from  those  obtained  in  other 
exijeriments  in  which  the  .serum  was  tested  only  after  the  animal  had 
been  immunized. 

The  undiluted  serum  from  this  hog  before  injection  was  quite 
strongly  tmctericidal  for  the  Crawford  and  the  F.  26  strains,  but  it 


EFFECTS   OF    DILUTION    UPON    BACTERIOLYTIC    POWER. 


43 


was  at  most  merely  inhibitor}'  for  the  G.  P.  4692  strain.  After  the 
second  and  third  injections  the  serum  seems  to  have  lost  all  potency 
for  all  three  organisms,  at  least  when  undiluted.  But  after  the  fourth 
injection  the  serum  seems  to  have  regained  its  potency  both  when 
undiluted  and  in  certain  of  the  diluticJns. 

Table  XXVIII. — Effect  of  dilution  upon  immuve  hog  serum  as  compared  with  the  effect 
of  dilution  upon  the  normal  serum.     Hog  1835,  injected  with  Crawford  culture. 


Culture 

used  to  test 

Time  blood  was  drawn 

Number  of 

bacteria 
introduced 
per  1  c.  c. 

Number  of  bacteria  In  serum  after  24 
hours. 

Number 
of  bac- 
teria in 
checksalt 
solution. 

bacterio- 
lysis. 

relative  to  injection. 

Undiluted. 

Diluted 
ItolO. 

Diluted 
ItolOO. 

Diluted 
1  to  1,000. 

Crawford  . . 

F.  26 

G.  P.  4692.. 

1  hour  after  injection  .. 

1  day  after  injection 

1  hour  after  injection  .. 

1  day  after  injection 

1  hour  after  injection  .. 
1  day  after  injection 

5,860 
3,200 
16,600 
6,900 
9,600 
4,600 

18,200 
71,100 

00 

0 
31,000 
3,600 

0 

5,200 

110,400 

40,400 

7,200 

96,000 

33.600 
11,000 

00 

28,000 
10,700 

a  oD 

GO 
00 

00 

oe 
00 

00 

102,200 

00 

00 

1,280? 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 

In  the  experiment  of  which  Table  XXVIII  gives  the  summary  the 
hog  was  injected  intravenously  with  a  dilute  suspension  of  the  Craw- 
ford culture.  Two  injections  of  the  organism  were  given  on  two  con- 
secutive days,  and  blood  was  drawn  from  the  animal  one  hour  and 
twenty-four  hours  after  the  last  injection.  No  blood  was  drawn 
before  injection  in  this  case; 

There  seems  to  have  been  no  difference  between  the  behavior  of  the 
blood  on  dilution  and  that  of  ordinary  normal  blood  on  dilution. 
Omitting  one  or  two  unimportant  discrepancies,  the  serum  lost  more 
and  more  in  potency  upon  further  and  further  dilution. 

Table  XXIX. — Effect  of  dilution  upon  immune  hog  serum  as  compared  with  the  effect  of 
dilution  upon  the  normal  serun..     Hog  1809,  injected  with  culture  G.  P. 


Culture 
used  to  test 

Time  blood  was  drawn 
relative  to  injection. 

Number  of 
bacteria 

introduced 
per  1  c.  c. 

Number  of  bacteria  in  serum  after  24 
hours. 

Number 
of  bac- 
teria in 
checksalt 
solution. 

bacterio- 
lysis. 

Undiluted. 

Diluted 
lto50. 

Diluted 
1  to  600. 

Diluted 
1  to  6,000. 

F.  26 

Crawford . . 

G.  P.  4692  .. 

Before  injection 

14  days  after  injection . . 

Before  injection 

14  days  after  injection.. 

Before  injection 

14  days  after  injection. . 

6,920 
2,400 
'       5,700 
13,000 
1,140 
7,300 

5,900 

1,800 

0 

5(;,  600 

10,900 

7,900 

109,200 

M68,000 

520 

64,000 

2,880 

69,200 

"    00 

6224,000 

00 

66,()00 
123,200 
625,900 

00 

6  00 

00 

6  05 

60,800 

6  00 

00 
00 

00 

00 

73,000 

00 

a  This  character  signifies  that  there  were  too  many  colonies  to  count. 
6  Diluted  in  the  proportion  of  1  to  10,  1  to  100,  1  to  1,000. 

In  the  experiment  summarized  in  Table  XXIX  the  hog  was  injected 
subcutaneously  with  .5  c.  c.  of  a  suspension  of  G.  P.  4692,  but  there 
seems  to  have  been  little  if  any  effect  produced  upon  the  bacteriolytic 
power  of  the  serum.  It  is  true  that  with  the  Crawford  strain  there  is 
some  evidence  of  loss  of  potency  in  the  undiluted  scrum,  and  of 
increased  potency  upon  dilution  in  the  serum  after  the  injection  of  the 
animal,  but  the  effects  on  the  whole  are  not  striking. 


44 


HArTERIOLYTIC    I»<>WKR   OF   BLOOD   8RRITM    OF    flOOS. 


Taulk  XXX. — AJffrt  of  dilution  upon  immuiu  hog  nertini  n*  romfttire  !  witii  tlif  tffirl 
o/ditutioit  upon  the  normal  aerum.     I  fog  ISOl,  injecUd  tvUh  (Vnwford  niihtre. 


Otiliurv 
UM<il  to  lent 

lyslii. 


r.». 


Crawford . 


(J.  P.  4«»2. 


Tlmv  IiIimmI  wKxilniwn  relative 
to  injei'tiuii. 


10  dayH  nftor  flnit  in  lection . . . . 

9  d»ys  nfter  ow'ond  injection. . 
14  rtuyH  iidtTiliinl  hijti'tloM... 

10  <li«v'<  nlU'T  t\vt  inicillon 

•  diiV!«  HfiiT  ftoi-oml  injfctlon.. 
It  dH>-«nft«Tthini  1nje«-tlnn... 

lOilnyxiifliT  tlrst  iiiW'ctlon 

C  clav  s  iificr  woHid  fiiJiTtlon. . 

1 1  <Im\-  itflrrtliini  injwtlon... 


Numberof  lwi'teriainm>niin*fUT'J4  ; 

Number 

of 
liict«ri« 

llOUfN.                                          ,  j 

intro- 
duced 
per  1  c.r. 

Undl- 

Diluted 

Dllnled 

Diluted  ' 

Inted. 

itoao. 

ItoAOO. 

lui.vooo. 

A.  400 

eeo 

24,400 

•o> 

1T2.000 

6,900 

4,180 

19.  MOO 

•^ 

» 

2,400 

60 

'>99.400 

6196.000 

fc  » 

840 

0 

640 

7,000 

I.46() 

.•V.700 

120 

19,000 

249.200 

« 

19.000 

0,220 

08,400 

h 12, 100 

66,4tti 

2,000 

S..VN) 

2,.'>40 

8.K20 

2,120 

1,140 

2.020 

2,8ttQ 

8,320 

7,600 

7.800 

7.800 

ft  11, 800 

t>8,100 

(•21.000  i 

.Slim- 

iHTOt 

bMie- 
rUin 
chocit 

unit 
HOlll- 

tion. 


oThls  charnctor  xlffniflcN  t)iat  there  were  too  many  colonlen  l«)  roiint. 
0  Diluted  1  ti>  10.  1  to  UiO.  1  to  1.000. 

The  h()j(  (IHOI)  in  the  experiment  shown  in  Table  XXX  wu-s  injected 
on  three  different  occusions  with  the  Crawford  or^nism.  No  test^ 
were  made  of  the  .serum  Ix^fore  the  ino<'uhition  of  the  animal,  but  there 
seems  little  or  no  evidence  of  the  reaction  usually  seen  in  the  .serum  of 
animals  injected  with  bacteria  in  the  tests  which  were  made  after  each 
injection.  The  .serum  in  most  of  the  tests-showed  lo.ss  of  ba<'teriolytic 
power  on  dilution,  bcluivintif  in  this  respect  like  normal  serum.  It  is 
true  that  with  the  homoloj^ous  strain— Crawford— the  .serum  ten  days 
after  the  first  injection  and  fourteen  days  after  the  third  injection 
appeared  to  l)e  somewhat  more  strongly  bacteriolytic  in  the  dilution 
of  1  to5,(K)0  and  1  to  1,000,  respectively,  than  in  the  le.ss  diluted  serum: 
but  on  the  whole  the  serum  .seemed  to  act  more  like  normal  than  like 
immune  .serum. 


Tablk  XXXI. — Kfffri  of  dilution  upon  immune  hog  xervm  an  rompared  uHh  the  rffert  of 
diltUion  ujnm  the  mtrmtU  $erum.     Hog  1836,  injected  tnf/j  cuUure  F.  S6. 


Culture 

UMed  to  text 

bacterio- 

lyalo. 


Crawford . . 


Time  blood  wan  drawn 
relative  to  injection. 


K.  26. 


G.  P.  4692. 


Before  injection 

1  hour  after  inlection  . 
8  dayx  iiftcr  injection.. 

Before  liijcrtiiin 

1  hour  after  iiiiiH'tlon  . 
Sdayw  uflcr  lnJe«'tlon.. 

Before  liijeotion 

1  hour  nf icr  inliTtion  . 
Sdayi  after  Injection.. 


Number  of 

iMcleria 
introduced 
per  1  c.  c. 


82.200 
82,200 

1.660 
80.400 
80.400 

5,000 

a^ooo 

86.000 
6.800 


houn. 


Ninnber  of  tiacteria  In  berum  after  24     J  Numlivr 

'  of  liicte- 
rill  in 
tieck  f«lt 
Muluiion. 


Diluted 

Diluted     bi.uted  c 

1  to  10. 

1  to  100. 

I  to  l.UUO. 

•  8,840 

820 

186,800 

6.1 

aft. 840 

240 

«B 

CB 

a» 

0 

161.400 

2^20O| 

0247.800 

117,600 

« 

3B    { 

a  » 

oc 

a» 

OD    1 

» 

96.000 

oe 

(» 

«47.400 

87,200 

aaH.000 

00 

«86,600 

8,400 

109. 200 

« 

OB 

U,600 

7^600 

286.600 

42,000 


8A8.600 


•>  Diluted  in  the  pn>p<trtion  of  I  |art  of  M'nitii  to  3  of  plivt'lotoKlral  ^alt  M>luiion. 
ftThlN  rhamcter  KiKnitieH  thai  (here  were  too  many  e«>louteii  to  <*«iunt. 

In  the  expi'riment  with  the  .serum  fi'om  the  bltxid  of  hog  1836.  Table 
XXXI,  the  animal  was  given  intravenously  .5  c.r.  of  a  dilute  l)ecf-broth 
suspension  of  an  agar  culture  of  F.  20  .strain.     One  day  after  the  first 


SUMMARY    OF    DILUTION    EXPERIMENTS.  45 

injection  the  animal  was  given  a  second  injection  of  the  same  amount 
of  the  same  strain.  The  blood  was  drawn  before  any  injection  was 
made,  one  hour  after  the  first  injection,  and  three  days  after  the  second 
injection. 

The  results  oDtained  with  the  serum  from  the  blood  of  this  hog,  at 
least  in  so  far  as  the  strains  G.  P.  4692  and  Crawford  are  concerned, 
are  in  striking  contrast  with  the  results  obtained  with  the  serum  from 
the  blood  of  hog  1801  (Table  XXX).  In  the  case  of  hog  1836  the  ani- 
mal was  injected  with  a  more  virulent  strain  than  was  the  case  with 
hog  1836,  and  it  is  possible  that  this  may  have  had  some  inliuenc© 
upon  the  results  in  the  two  cases.  But  aside  from  this  the  two  sera 
must  have  had  ditferent  potency  originally,  for  even  the  normal  serum 
of  the  blood  of  hog  1836,  before  the  animal  was  injected,  will  be  seen 
to  have  shown  increase  of  potency  upon  dilution;  the  dilution  of  1  to  10 
was  more  potent  than  the  dilution  of  1  to  4.  There  was  no  test  made  of 
the  undiluted  normal  serum  for  the  reason  that  not  enough  of  the  blood 
was  obtained  to  use  for  the  purpose.  This  increase  of  potency  on 
dilution  of  the  normal  serum  of  hog  1836  is  to  be  seen  particularly  in 
the  tests  with  the  Crawford  strain,  in  which  the  dilution  of  1  to  4 
showed  3,840  bacilli  per  1  c.  c.  whereas  the  dilution  of  1  to  10  showed 
only  320  bacilli  after  the  same  exposure — one  day  in  each  case — with 
the  same  number  of  organisms — 32,200  per  1  c.  c. — introduced.  This 
serum  in  other  words  seems  to  have  behaved  somewhat  like  immune 
serum  to  start  with,  and  this  behavior  seems  to  have  been  intensified 
by  the  injections,  as  will  be  seen  by  examining  the  table. 

SUMIVIARY   OF   THE    DILUTION    EXPERIMENTS. 

On  summing  up  the  results  of  the  experiments  made  with  normal 
and  inmiune  hog  serum  on  dilution,  it  will  be  noticed  that  the  normal 
serum  seems  to  differ  greatl}'  in  its  behavior  in  difierent  cases;  that 
while  it  sometimes  shows  marked  diminution  of  potency  upon  dilution 
it  sometimes  shows  increased  potency  on  dilution.  The  immune  hog 
serum  also  shows  variation  in  behavior. 

But  the  results  which  would  seem  to  be  of  especial  interest  are  those 
in  which  the  immune  serum  showed  strong  bacteriolytic  power  when 
undiluted,  and  lost  in  power  on  dilution  in  certain  proportions,  but 
gained  potency  upon  further  dilution.  In  these  cases  there  appeared 
to  be  a  combination  of  the  behavior  of  normal  and  of  immune  serum, 
as  this  is  usually  stated  to  occur. 

As  alreadj^  stated  above,  the  characteristic  behavior  of  immune 
serum  is  said  to  consist  of  the  formation  of  bacteriolytic  zones.  A 
typical  immune  scrum  reaction  consists  of  a  pro-killing  zone,  a  killing 
zone,  and  a  post-killing  zone  on  dilution.  In  the  pro-killing  zone, 
represented  by  the  undiluted  immune  serum,  there  is  no  bacteriolysis. 
In  the  killing  zone,  represented  by  one  or  more  dilutions,  there  is 


46  BA(rrERI«)LYTir    1»<>WKR    ok    HL<H)D   SKRUM    ok   H<W8. 

liactoriolysis.  In  the  ix>.st-killin^  zone,  repn.?Mont<Hl  l)y  furtlier  dilu- 
tion than  thi'  killinjr  /one,  theiv  h  apiin  hu'k  of  lnict<>rioIysi8.  Hut  in 
th<»  ivsults  at  present  und«»r  oonsiiitM'tition  the  serum  showotl  ii  killing 
zone  tt)  hegin  within  th«'  undiluted  seruin.thus  iinitntitit;  to  tliis«'xtont 
the  nonnal  scnim. 

The  |)eeulinr  Indiavior  just  alluded  to  wa.s  to  Ik'  .st?en  in  several 
instances  jjiven  in  the  precedinj;  tahles.  Perlmps  the  most  striking 
exiimples  are  furnished  by  the  Inrhaviorof  the  immune  sera  from  hogs 
171>S  and  isos  (see  Tahle  XXIV).  In  these  sera  with  the  Cniwford 
culture  there  was  strong  l>acteriol3'sis  shown  in  both  ea.ses  undiluted. 
The  1H(»8  wa«  still  j-trongly  potent  in  the  1  to  50 dilution,  but  the  1798 
serum  was  much  less  potent  in  this  dilution  than  when  undiluted. 
IJoth  sera  seem  to  have  lost  all  |X)teney  when  diluted  in  the  pro|X)ition 
of  I  to  500.  But  the  point  to  be  spt»cially  noted  is  that  they  l)oth 
seem  to  have  gained  greatly  in  the  dilution  of  1  to  5,000;  particularly 
is  this  the  cjuse  with  the  17V»8  serum. 

For  the  present  it  is  merely  necessar\'  to  call  attention  to  these 
results:  they  will  be  tiikcn  up  and  discussed  in  another  connection  fur- 
ther along  in  the  present  paper. 

GENERAL  DISCUSSION. 

Judging  from  the  results  of  the  abov«^  experiments  as  a  whole,  it 
seems  apparent  that  the  blood  serum  of  hogs  has  but  feeble  bactericidal 
potenc}'  for  B.  c/io/^ne  hu/m,  at  least  for  the  more  strongly  pathogenic 
strains.  This  would  naturally  lead  to  the  presumption  that  the  known 
susceptibilit}'  of  hogs  to  infection  by  intravenous  injection  was  due  to 
this  cause,  ))ut  it  does  not  seem  as  3'et  settled  that  the  susceptibility 
of  an  animal  in  general  is  in  direct  j)rop<)rtion  to  the  want  of  bjicteri- 
cidal  ix)wer  of  its  blood.  In  fact,  in  some  instances,  as  is  well  known, 
quite  the  contrary  has  been  shown  to  be  the  case.  Nuttall,  Buchner, 
and  many  othei*s  have  shown  that  rabbit  serum  in  test  tulH\s  will  kill 
large  numbei's  of  anthrax  bacilli,  and  yet  a  comparatively  small  num- 
ber of  these  organisms  intro<luced  beneath  the  skin  of  a  rabbit  is  cer- 
tainly fatal  to  the  animal.  Guinea-pig  serum,  as  recently  shown  by 
Buxton,*  may  l^e  expi»cted  to  kill  approximately  l,(MM),o<Hj  typhoid 
bacilli  per  1  c.  c,  and  yet  the  injection  of  a  relatively  small  amount 
of  this  organism  intrajM»ritoneally  is  certainly  fatal  for  guinea  pigs. 
In  spite  of  this,  however,  as  Wolff'"  has  |)ointed  out,  it  is  prohibly  all 
a  matter  of  dosjige,  and  Iwicteriolysis  is  an  efficient  means  of  defense 
where  the  numl>er  of  Iwicteria  concerned  is  not  large.  On  the  con- 
trary, where  the  numl^er  of  Iwcteria  exceeds  certain  limits,  Imcteri- 
olysis  is  not  only  not  a  nu»ans  of  defense,  but  it  actually  lil»erates  the 
disease-producing  sub.stances — the  endotoxins — from  the  Ixxiies  of  the 
bacteria. 


GENERAL    DISCUSSION.  47 

Pfeiffer"  was  the  first  to  give  this  name,  endotoxins,  to  the  poison- 
ous substances  which  are  present  in  the  bodies  of  the  bacteria  of 
certain  species,  and  it  is  now  quite  generally  the  opinion  that  it  is  the 
liberation  of  these  that  brings  about  disease  in  infection  with  these 
bacteria.  So  long  as  bacteria  of  this  kind  remain  intact,  they  are 
thought  by  those  holding  this  view  to  be  incapable  of  producing  dis- 
ease. Mechanical  injury  and  other  harmful  effects  formerly  attributed 
to  the  action  of  bacteria  are  now  thought  by  many  not  to  exist. 

The  effects  of  inoculations  of  hogs  with  cultures  of  B.  cholerse  suis^ 
and  the  conclusions  to  be  drawn  from  these  experiments  are  on  the 
whole  in  harmon}^  with  those  derived  from  experiments  with  various 
bacteria  injected  into  rabbits,  guinea  pigs,  and  other  animals.  Thus 
Loeffler  and  Abel*^  found  that  undiluted  immune  blood  serum  from 
dogs  failed  to  protect  guinea  pigs  from  injections  of  virulent  colon 
bacilli.  Neisser  and  Wechsberg"  obtained  analogous  results  with 
immune  rabbit  serum  in  test  tubes,  the  undiluted  immune  serum  failing 
to  destroy  the  bacteria  of  the  kind  used  to  produce  the  immune  serum. 
Buxton,*  as  already  quoted,  found  that  the  undiluted  serum  of  rabbits 
injected  with  cultures  of  typhoid  or  parat}' phoid  bacilli  behaved  in  a 
similar  manner.  It  was  to  be  expected,  therefore,  that  the  serum  of 
hogs  injected  with  cultures  of  B.  cJiolerse  suis  would  show  loss  of 
bacteriolytic  power  in  undiluted  sferum.  The  behavior  of  the  diluted 
serum  will  be  considered  below. 

In  the  foregoing  experiments  with  the  serum  from  the  blood  of 
hogs  injected  with  cultures  of  B.  cholerai  suis,  it  appears  that 
the  effect  of  the  tirst  injection  was  sometimes,  as  stated,  to  deprive 
the  undiluted  serum  of  its  power  to  kill  B.  ckoler^as  suis.  It  is  true 
that  this  behavior  was  frequently  lacking.  But  if  the  results  after 
several  mjections  are  examined,  it  will  be  found  that  the  undiluted 
serum  frequently  regains  its  power.  This  is  noticeable  in  a  number 
of  cases  if  not  in  all,  and  it  perhaps  accounts  for  the  experience  of  so 
many  observers  that  animals  frequently  die  from  injections  of  com- 
paratively small  doses  after  having  been  treated  with  carefully  graded 
amounts  of  culture.  Thus  Wolff"  ^"^  found  that  strong,  healthy  rabbits, 
instead  of  becoming  more  and  more  accustomed  to  injections  of 
cultures,  withstood  these  less  and  less  readily,  even  without  increasing 
the  dose;  that  after  remaining  apparently  unaffected  by  previous 
injections,  they  often  die  suddenly  on  the  inoculation  being  repeated 
with  the  same  sized  dose.  This  supersensitiveness  on  repeated  in- 
jections has  often  been  observed  in  various  animals  and  with  various 
bacteria.  It  has  also  been  met  with  in  the  course  of  the  present 
experiments  with  hogs.  In  fact,  it  does  not  seem  so  difficult  to  grade 
the  first  dose  so  as  to  avoid  killing  the  animal  as  it  is  to  strike  upon 
the  proper  subsequent  doses.  The  return  of  bacteriol^^tic  properties 
in  the  serum  following  further  injection  of  animals  may  account  for 


48  BACTERI(>LYTIC   PoWKR   OF    HL<M)D   SKRCM    OF    HoaS. 

this  result,  the  first  injections  deprivinji;  the  serum  of  tlie  liaeteriolytic 
power  and  thus  preventing  the  lilieration  of  endotoxin  and  its  con- 
sequenees,  and  the  suhse<|uei)t  injections  produeinji^  a  return  of 
bacteriolytic  |K)\ver  and  aiusin^  the  lilH>ration  of  the  endotoxin. 

It  would  seem  perhaps  ditticult  to  reconcile  the  fact  that  the  serum 
iKH'omes  more  |X)tent  for  the  prote«-tion  of  animals  other  than  the 
individual  which  prcxluces  it,  and  that  yet  from  the  very  fact  of  its 
bacteriolytic  power  it  should  l)e  injurious  to  the  animal  furnisbin);  it. 
It  would  ap|>ear  |>aradoxical  to  lind  that  scrum  obtained  from  an  ani- 
mal possessed  the  property  of  protcctinj^  other  animals,  and,  at  the 
same  time,  not  onl}'  failing  to  protect  the  animal  furnishing  it,  but 
actually  increasing  the  susceptibility  of  this  animal.  Mere  again 
Woltf  offers  the  explanation  that  it  is  a  matter  of  dosage.  If  the 
numl>er  of  Imcteria  injected  into  the  animal  furnishing  the  serum,  or 
into  the  animal  which  has  been  given  a  ])rotective  dose  of  the  serum, 
is  not  so  large  that  on  being  disintegrated  the  amount  of  toxin  is  suf- 
ficiently large  to  cause  disease,  the  animal  escapes.  There  are  appar- 
ent ditficulties  in  the  way  of  this  explanation,  but  it  is,  perhaps, 
nevertheless  not  without  weight. 

In  this  connection  the  results  obtained  by  Elischer  and  Kentzler* 
on  comparing  the  bactericidal  jx)wer  of  unheated  immune  serum — 
i.  e.,  immune  serum  containing  its  own  pro|)er  complement — with  the 
same  serum  heated  and  reactivated  by  the  addition  of  fresh  rabbit 
serum  would  seem  to  apply.  The  latter  was  found  to  be  more  iK)tent 
than  the  former,  and  this  would  seem  in  a  way  to  offer  an  explanation 
of  the  protective  power  of  immune  serum  for  an  animal  into  which  it 
is  injected  on  the  one  hand  and  of  the  failure  of  such  protective  prop- 
erties of  the  same  serum  for  the  animal  from  which  it  was  obtained 
on  the  other.  Elischer  and  Kentzler  interpret  their  results  as  indi- 
cating that  immune  serum  contains  an  insufficient  amount  of  com- 
plement for  the  full  development  of  its  bactericidal  properties.  So 
in  other  cases,  the  amount  of  complement  in  immune  serum  may  be 
insufficient  to  enable  the  serum  to  protect  the  animal  from  which  it 
was  obtained,  while  it  would  find  sufficient  complement  in  the  serum 
of  a  fresh  animal.  To  state  this  view  briefly,  the  excess  of  aml)ocep- 
tors  in  immune  serum  finds  the  requisite  amount  of  complement  in 
the  normal  serum  of  uninfected  animals. 

When  the  l>ehavior  of  hog  serum  on  dilution  is  considered,  the 
exi)lanation  afforded  ]»y  the  theor}'  of  complement  diversion  sfM^ms 
applicable,  not  only  for  the  behavoir  of  imnume  serum  in  which  there 
was  shown  often  to  be  increased  l>actericidal  potency  on  dilution,  but 
also  in  the  case  of  normal  serum,  as  it  will  be  attempted  to  show. 

It  will  be  recalled  from  the  statements  in  regard  to  the  theory  of 
complement  diversion  given  on  page  13  that  in  accordance  with  the 


GENERAL    DISCUSSION.  49 

views  of  the  Ehrlich  school  generally  it  is  held  by  Neisser  and 
Wechsberg,  the  originators  of  the  theory,  that  bacteriolysis  is  due 
to  the  action  of  a  substance  called  complement,  which  is  present  in 
normal  and  in  immune  serum,  but  that  this  substance  can  act  on  the 
bacteria  only  when  it  becomes  united  to  these  through  the  medium  of 
certain  bodies  called  amboceptors.  The  complements  are  held  to  be 
incapable  of  uniting  directly  with  the  bacteria.  Now,  according  to 
the  theory  of  complement  diverson,  if  there  are  more  amboceptors 
than  complements  present  in  any  given  serum,  all  the  complements 
which  are  present  unite  with  a  corresponding  number  of  amboceptors, 
and  if  any  bacteria  are  introduced  into  the  serum  they  unite  with  the 
free  amboceptors  in  preference  to  uniting  with  those  having  comple- 
ments attached.  In  this  way  the  complements  become  diverted  from 
the  bacteria  by  the  excess  of  amboceptors.  It  will  also  be  recalled 
that  while  there  are  amboceptors  in  normal  serum,  these  become 
increased  when  the  animal  from  which  the  serum  is  obtained  is 
injected  with  bacteria.  The  amboceptors  formed  by  these  injections 
are  specific;  that  is  to  say,  an  animal  injected  with  B,  cholerse  suis 
develops  amboceptors  in  the  serum  which  are  capable  of  uniting  with 
B.  cholerse  suis  only.  Reference  to  figure  4  (page  51)  will  serve 
to  elucidate  whatever  may  be  obscure  in  the  above  explanation  of  the 
theory  of  complement  diversion. 

Bearing  in  mind  that  normal  serum  contains  a  relatively  large 
amount  of  complement  and  a  relatively  small  number  of  ambocep- 
tors, it  is  evident  that  by  introducing  normal  serum  into  immune  serum 
it  is  possible  to  increase  the  amount  of  complement  without  adding 
materially  to  the  number  amboceptors.  In  fact,  by  making  a  series 
of  dilutions  of  immune  serum  and  adding  to  each  lot  in  the  series  a 
definite  amount  of  normal  serum,  it  is  possible  to  obtain  a  set  of  dilu- 
tions containing  various  amounts  of  amboceptors  and  approximately 
the  same  amount  of  complement.  Evidently  if  the  immune  serum  is 
merely  diluted  and  no  normal  serum  is  added,  the  complements 
present  will  be  diluted  in  the  same  proportion  as  the  amboceptors. 

If  the  amount  of  complement  is  kept  constant  in  the  way  above 
described,  by  the  addition  of  a  constant  amount  of  normal  serum,  it  is 
evident  that  in  some  of  the  dilutions  of  the  immune  serum  the  ambo- 
ceptors will  have  enough  or  more  than  enough  complements  to  com- 
bine with  the  amboceptors,  and  that  in  such  cases  the  conditions 
necessary  for  bacteriolysis  will  be  fulfilled — the  amboceptors  with  the 
complements  attached  will  unite  with  the  bacteria.  In  other  words, 
the  undiluted  immune  serum,  having  an  excess  of  amboceptors  over 
complements,  fails  to  show  bacteriolysis,  while  the  diluted  immune 
serum  with  complements  added,  having  no  excess  of  amboceptors 
over  complements,  causes  bacteriolysis. 


60  BAOTKRIOLYTK^    1*«)WKR    OK    KL<H)D   SERUM    OF    HOOS. 

Buxton*  has  misod  the  ohjoctum  t«»  tlio  thoorv  that  ii  ram  m « ..uui 
for  those  ni.s««s  only  in  whirh  the  iinuMint  of  complement  is  kept  con- 
stant in  thi>  (iiliitions  of  immune  serum,  while  the  lunlMM-eptors  nlone 
nrv  diminished.  IIo  miikes  the  point  thiit  in  e\{)eriments  sueh  us  arc 
recoitied  in  the  present  |m|HM',  in  which  andMJceptor.s  and  comple- 
ments arc  diluted  to  an  cipial  extent,  the  theory  of  <'omplcment  diver- 
sion is  inapplicable;  and  since  the  pnx^ess  is  probjibly  the  same  in  l>oth 
cases,  Inith  where  the  complement  is  kept  to  a  constant  amount  b}' 
heating  the  s(>rum  and  adding  a  detinite  ({uantity  of  normal  serum  on 
the  one  hand  and  where  the  serum  is  not  heated  and  has  no  normal 
serum  added  on  the  other  hand,  the  theory  as  a  whoh'  is  not  tenable. 
Valid  as  this  objection  appears  from  Buxton's  presentation,  it  appears 
on  further  consideration  as  scarcely  to  be  insuperable,  for  the  theory 
seems  to  afford  a  satisfactory  explanation  in  every  ca.se  where  the 
amlK)ceptors  are  in  excess  of  the  complements,  and,  j^ranting  a  cer- 
tain proportion  l>etween  these  in  the  undiluted  .serum,  it  is  not  neces- 
sary for  the  complement  to  be  present  in  an  equal  amount  in  the  undi- 
luted senim  and  in  the  dilutions  for  the  occurrence  of  the  diversion 
of  the  conjplement  to  take  place.  All  that  the  theor}'  would  seem  to 
demand  is  that  the  undiluted  serum  contain  an  insufficient  amount  of 
complement  to  satisfy  the  amboceptors  which  are  free  and  those  which 
are  atUiched  to  the  bacteria.  IJacteriolysis  will  take  place  just  in  pro- 
l>ortion  to  the  jmucity  of  the  free  aniboceptors,  for,  according  to  the 
theory,  those  Imcteria  which  are  not  coupled  to  free  anilx>ceptors 
are  liable  to  bacteriolysis  by  becoming  attached  to  the  amboceptor- 
complement  combination.s. 

In  figure  4  it  is  attempted  to  show  that  comp.ement  diversion  may 
well  account  for  those  ca.ses  in  which  the  complement  is  diluted  in 
the  same  proportion  a.s  the  amboceptors.  This  point  could  be  more 
strikingly  shown  if  a  larger  number  of  amboceptors  and  complements 
could  Ik?  taken,  and  a  much  greater  relative  difference  could  Ik»  made 
in  the  diagnim  between  the  nura>)er  of  amlKKjeptors  and  of  comple- 
mentu,  but  for  obvious  rea.sons  it  is  impracticable  to  do  this.  Thus,  if 
it  were  feasible  to  show  100  amboceptors  and  100,000  amlxK'eptor- 
complement  combinations  in  1  c.  c.  of  undiluted  .senim.  it  could  l>e 
demonstrated  that  100  bacteria  introduced  would  all  escaixj  bacterio- 
lysis in  this  serum,  only  10  bjicteria  would  escape  in  1  c.  c.  of  a  dilu- 
tion of  I  to  10  of  the  same  serum,  only  1  l>act<>rium  would  e.sca|)e  in  1 
c.  c.  of  a  dilution  of  1  to  100,  and  none  would  escape  in  1  c.  c.  of  a 
dilution  of  1  to  1,0(K).  In  other  words,  theoretically  in  serum  in  which 
the  al)ovo  conditions  prevailed,  taking  1  c.  c.  in  every  case  and  intro- 
ducing 100  bacteria,  there  would  be  no  bacteriolysis  in  the  undiluted 
serum,  but  complete  bacteriolysis  in  the  same  serum  in  the  1  to  1,000 
dilution. 


GENERAL    DISCUSSION. 


51 


In  figure  4  the  amboceptors  are  represented  by  the  parts  marked 
a,  the  complements  by  the  parts  marked  Ji\  and  the  bacteria  by  the 
parts  marked  h.  No.  1  is  intended  to  represent  the  undiluted  serum, 
and  all  except  two  of  the  amboceptors  are  shown  as  having  diverted 
a  corresponding  number  of  complements,  while  the  two  amboceptors, 
which  are  not  united  to  complements,  and  which  represent  the  excess 
of  these  bodies  over  complements,  are  united  to  a  corresponding  num- 
ber of  bacteria.  Evidently  in  such  a  case  there  would  be  no  bacte- 
riolysis, since  all  the  complements  have  been  diverted  from  the 
bacteria.     In  No.  2  half  the  amount  of  the  serum,  as  in  the  first  case — 


Fig.  4.— Diversion  of  complement  in  immune  serum,  not  heated,  and  vithout  the  addition  of 

foreign  complement. 

that  is  to  say,  1  c.  c.  of  a  1  to  1  dilution — is  represented,  with  the 
same  number  of  bacteria  added  as  in  the  undiluted  serum  represented 
by  No.  1.  Under  the  conditions  represented  by  No.  2  it  is  evident 
that  one-half  of  the  bacteria  would  be  killed.  In  No.  3  is  represented 
1  c.  c.  of  a  dilution  in  which  0.25  c.  c.  of  the  original  serum  is  contained. 
In  this  case  all  the  bacteria  would  be  killed,  since  there  are  no  longer 
any  free. amboceptors  to  unite  with  the  bacteria,  or,  at  any  rate,  there 
would  not  be  enough  of  the  amboceptors  to  divert  the  complement  in 
all  of  the  four  portions  into  which  it  is  assumed  the  serum  is  divided. 
If  the  amboceptors  are  to  be  regarded  as  chemical  substances  in  solu- 
tion, then  there  would  not  be  enough  in  Any  one  of  the  four  portions 
to  cause  diversion  of  the  complement.     If,  on  the  other  hand,  the 


52  BACTERIOLYTIC    F<»WKR   Of   BLcK>D   SERUM    ()K   H()G8. 

anilKM'optors  nro  l)odios  in  the  strict  sense,  then  it  in  evident  that  two 
of  the  four  |X)rtions  would  show  Iwiet^M'iolyHi.H  of  one-half  of  the  l>ac- 
teria,  while  the  other  two  would  show  <'oniplete  Imctrriolysis,  a>iHum- 
injf  uniform  distribution  of  the  amboceptors.  In  either  case  the 
diluted  serum  would  be  more  powerfully  Vmctericidal  than  the  undi- 
lute<l  senim.  It  will  be  observed  that  the  complement  is  not  ki'pt 
at  a  constant  amount  in  the  dilutions  represented  by  the  diagrams, 
but  that  it  is  diluted  sinmltaneously  and  to  a  like  degree  with  the 
amlKweptors. 

From  what  has  been  said  above  it  would  appear  that  diversion  of 
t^mplement  seems  a  reasonable  explanation  of  the  phenomena  which 
are  observed  in  unheated  unreactivated  serum,  and  it  therefore  seems 
to  overcome  Huxton's  objection  to  the  application  of  the  theorj'  to  the 
phenomena  observed  under  such  conditions. 

It  is  aside  from  the  purpose  of  the  present  article  to  try  to  meet  in 
general  objections  to  the  theory  of  complement  diversion,  or  to  discuss 
this  theory  apart  from  whatever  bearing  it  may  have  ujKjn  the  present 
experiments;  but  incidentally  the  objection  raised  by  Buxton  of  bring- 
ing in  accord  with  the  theory  of  complement  diversion  the  fa<-t  that 
Asiatic-cholera  spirilla  are  killed  about  as  readily  by  typhoid  and  jjara- 
t3'phoid  immune  serum  as  by  normal  serum  may  perhaps  be  overcome 
by  consideration  of  the  fact  that  the  injection  of  an  animal  with  any 
one  kind  of  l)acterium  probabl}'  has  no  effect  upon  the  amlxx-eptor- 
complement  combinations  for  any  other  kinds  of  l)acteria  which  are 
present  in  the  serum  of  the  animal  undergoing  inoculation.  The  effect 
is  probably  very  sharply  specitic,  even  going  so  far  in  some  cases  as  to 
affect  different  strains  of  the  same  organism,  as  is  shown  by  the  results 
of  some  of  the  experiments  in  the  present  paper.  It  seems  not  at  all 
inconceivable,  at  least,  that  an  animal  injected  with  any  given  kind  of 
Imcterium  might  well  be  deprived  of  the  power  of  killing  the  kind 
of  Imcterium  with  which  it  was  injected,  owing  to  the  excessive  pro- 
duction of  specific  amboceptors,  without  interfering  with  the  ambo- 
ceptor-complement  combinations  for  any  other  bacteria  which  may  be 
originally  present. 

In  view  of  the  very  specitic  reaction  which  takes  place  on  injecting 
an  animal  with  bacterial  cultures,  there  would  really  seem  to  be  no 
good  reason  why  immune  serum  should  act  differently  from  normal 
serum  for  anj'  but  the  homologous  organism.  The  amlK)ceptors  which 
are  newly  formed  in  consequence  of  the  injections  could  not  reasonabl}- 
be  supposed  to  have  the  power  of  depriving  the  amboceptors  originally 
present  of  the  complements  to  which  they  are  attached.  If  it  is  true,  as 
assumed,  that  in  normal  serum  there  are  amboceptor-complement  com- 
binations which  will  destroy  B.  choler»  »ui\  and  in  the  same  serum 
anilx)ceptor-complement  combinations  which  destroy  B.  tt/pho8w<^  the 
injection  of  the  animal  with  cultures  of  B.  (*/<o^a;«ui«  would  evidently 


GENERAL    DISCUSSION,  53 

increase  the  amboceptors  for  this  organism,  and  these  newly  formed 
amboceptors  would  have  the  power  of  uniting  with  any  free  comple- 
ment, but  not  with  any  complement  already  united  to  the  B.  typhosm 
amboceptors.  So  that  while  the  newly  formed  amboceptors  would 
and  probably  do  find  free  complements  with  which  to  unite,  they  could 
hardly  be  expected  to  tear  loose  the  bonds  between  the  original  ambo- 
ceptors and  complements. 

As  has  been  pointed  out  by  Neisser  and  Wechsberg  in  their  experi- 
ments, the  increase  of  bacteriolysis  usually  observed  on  the  dilution  of 
immune  serum  does  not  appear  to  be  explicable  by  the  conception  of 
Bordet  that  the  immune  body — the  amboceptor — is  merely  a  sensitizing 
agent.  For  if  the  amboceptor  were  in  reality  merely  a  "substance 
sensibilisatrice "  it  is  not  likely  that  immune  serum  would  be  more 
active  when  dilute  than  when  concentrated.  At  least  it  would  be  only 
reasonable  to  presume  that  a  reagent  possessing  the  properties  ascribed 
to  the  sensibilisatrice  by  Bordet  and  his  followers  would  lose  rather 
than  gain  in  power  upon  dilution,  and  that  a  given  amount  of  the 
undiluted  sensibilisatrice  would  be  more  potent  than  the  same  amount 
of  the  diluted  substance,  as  is  the  case  with  other  reagents.  On  the 
other  hand,  Ehrlich's  conception  of  the  amboceptor  affords  a  satis- 
factory explanation  of  the  behavior  of  immune  serum  upon  dilution,  as 
has  been  shown  in  the  above  discussion  of  the  theory  of  complement 
diversion. 

The  results  obtained  in  the  present  investigation  in  which  the  hog 
serum  appeared  to  increase  in  potency  upon  dilution  seem  best  ex- 
plained by  the  theory  of  complement  diversion.  But  in  these  experi- 
ments, as  in  all  others  of  like  nature,  it  would  seem  possible  that  the 
process  of  bacteriolysis  or  the  failure  of  bacteriolysis  may  at  least  be 
complicated  b}^  the  formation  of  antibacteriolysins  on  the  injection  of 
the  hogs  or  by  the  presence  of  antilysins  normally  present  in  the 
serum.  Opinions  are. divided  upon  the  question  as  to  whether  there 
are  such  antilysins  present  in  normal  or  in  immune  serum.  But  if 
there  should  be  such  antibodies  present,  it  is  reasonable  to  suppose 
that  these  would  be  weakened  in  power  on  diluting  the  serum,  and 
that  consequently  such  serum  would  be  more  potent  when  diluted  than 
when  undiluted,  unless  the  lysine  and  antilysins  should  be  diluted  in 
like  proportions. 

There  seems  to  be  no  difference  of  opinion  in  regard  to  the  develop- 
ment of  qualities  antagonistic  to  bacteriolysis  in  serum  under  certain 
circumstances,  but  the  matter  in  dispute  is  whether  this  antagonistic 
property  is  due  to  special  antilysins  or  whether  it  is  due  to  the  pres- 
ence of  nonspecific,  normal  amboceptors  or  of  superabundance  of 
immune  amboceptors  which  absorb  the  complement,  and  in  this  way 
prevent  bacteriolysis. 
•   Pfeift'er  and  Friedberger,^®  who  were  the  first  to  detect  this  antago- 


54  BACTKRIOLYTir    POWER    OF    BLOOD   AKRUM    OF    H008. 

nistic*  action  on  tho  part  of  normal  serum,  hold  that  under  t>ertain  cir- 
fumstancoH  specific  antilMxlics  are  formed,  which  combine  with  the 
complcnuMit.  In  other  words,  thoy  ivjjard  the  antilysins  as  anticom- 
plemcnts.  Their  cxjM'rimcnts  consist  of  triturating  norniul  scrum 
with  Iwt'teria  and  centrifujfali/injf.  Under  such  circumstances  the 
supernatant  serum  ac(|uires  the  ])ro|)erty  of  neutmli/injf  the  })actcrio- 
lytic  |)ower  of  inimiinc  serum.  Tims  normal  serum,  which  in  itself 
exerts  noantihacteriolytic  |x>wer,  Iwcomesantibacteriolytic  for  cholera 
inmnnie  serum  when  it  is  tritumted  with  choleni  hacilli  and  centrifu- 
jfalized  or  tiltered  through  a  l>acteria-proof  tilter, 

(Jay'*  attributes  antilytic  projx^rties  developed  in  this  way  to  the 
formation  of  sjM^citic  precipitines.  He  finds  that  only  when  there  is  a 
sp«'cific  precipitation  is  there  any  antilytic  pro|)erty  shown  hy  hemo- 
lytic sera,  and  since  hemolysis  and  bacteriolysis  are  prolwibly  identical 
processes,  the  same  statement  would  apply  to  the  latter  as  well  as  to 
the  former. 

(iay's  view  has  been  called  into  question  by  Sachs,"  who  attributes 
the  antilytic  pro|XM*ties  of  serum  to  normal,  nonspecific  aml)oceptors 
which  he  regards  as  acting  as  neutralizers  of  the  complement.  The 
otdy  difference  l>etween  the  explanation  offered  by  Pfeiffer  and  Fried- 
berger  on  the  one  hand  and  Sachs  on  the  other  is  that  while  the  former 
look  upon  the  antilysins  formed  in  their  experiments  as  newly  formed 
bodies  derived  in  part  from  the  bacteria,  Sachs  contends  that  the 
antagonistic  Iwdies  are  present  in  the  serum  from  the  start.  He 
states,  at  least,  that  this  is  the  only  point  in  which  he  takes  issue  with 
Pfeiffer  and  Friedberger  on  the  matter  of  the  formation  of  antilysins 
for  hemolytic  seruin. 

Lipstein,"  on  <"omparing  strongly  agglutinating  imnmne  seni,  found 
that  while  Imth  the  specimens  examined  possessed  equal  agglutinating 
power  only  one  of  them  showed  the  phenomenon  of  complement  diver- 
sion— that  is  to  say,  antibacteriolytic  properties.  He  was  also  unable 
to  discover  any  constituent  of  the  normal  seriuu  which  showed  diver- 
sion of  complement,  but  on  the  contrary  he  found  this  phenomenon 
to  take  place  in  immune  serum  only.  In  other  words,  the  antagonistic 
action  of  serum  to  Imcteriolysis  he  attributes  .solely  to  the  diversion  of 
complenjcnt  by  the  s|K»cific,  inunune  amlKK-eptors. 

In  spite  of  this  conflict  of  authority,  it  would  .seem  at  least  that  the 
Neisser-Wechsberg  phenomenon  pemiits  of  easiest  explanation  by  the 
theory  of  complement  diversion.  For  if  the  failure  of  the  undiluted 
inunune  .senun  to  produce  bacteriolysis  were  due  to  the  presence  of 
antilysins,  it  would  l>e  less  potent  uixm  diluting  the  .serum,  it  is  true, 
and  consejuiently  exert  less  |>ower  u|)on  the  Imctcriolysins  when  diluted 
than  when  undiluted,  but  the  relative  effect  would  remain  the  .same. 
It  is  nevertheless  prolmble  that  if  Imctcriolysins  are  present  in  immune 
aerum  they  complicate  the  process. 


GENERAL    DIHCUS8I0N.  55 

While  the  theory  of  complement  diversion  seems  to  have  been  used 
by  all  investigators  to  explain  the  bacteriolytic  phenomena  of  immune 
sera  onl}^,  it  would  not  seem  inapplicable  to  those  which  take  place  in 
normal  serum  as  well.  The  theory  seems  at  least  to  explain  the 
behavior  of  the  normal  serum  of  hogs  upon  dilution  in  the  present 
investigation.  Even  in  those  cases  where  the  normal  serum  showed 
diminution  of  potency  on  dilution,  the  diminution  was  not  in  direct 
proportion  to  the  dilution,  as  has  been  previously  noted;  but  on  the 
contrary  the  diluted  normal  serum  acts  more  powerfullj',  relatively, 
than  the  undiluted  normal  serum.  This  relatively  greater  potenc}'  of 
the  diluted  normal  serum  o||ppr  the  undiluted  may  be  accounted  for 
by  the  theory  of  complement  diversion,  if  it  is  assumed  that  there  is 
an  excess  of  amboceptors  over  complements  in  the  normal  serum  of 
hogs  to  start  with,  as  certain  considerations  will  show. 

Difficulties  in  the  way  of  the  acceptance  of  the  explanation  just 
offered  are  of  course  very  obvious.  In  the  first  place,  normal  serum 
generally  has  been  found  to  contain  an  excess  of  complement  over 
amboceptors;  in  fact,  in  adding  normal  serum  to  heated  immune 
serum,  this  is  done  for  the  purpose  of  increasing  the  amount  of  com- 
plement without  adding  to  the  amboceptors.  In  heating  immune 
serum  it  is  the  complement  which  is  rendered  for  the  time  inactive,  as 
it  is  called,  and  the  addition  of  fresh  serum  reactivates  the  immune 
serum  by  means  of  the  new  complement  contained.  The  addition  of 
fresh  serum  to  the  heated  t3^phoid  immune  serum  of  a  rabbit  reacti- 
vates the  immune  serum.  On  account  of  the  large  amount,  or  of  the 
great  number,  of  complements  contained  in  the  serum  from  the  blood 
of  a  normal  horse  this  can  be  used  to  reactivate  several  different  kinds 
of  immune  sera.  Then  again,  as  stated  above,  in  the  process  of  im- 
munization, the  newly  formed  amboceptors  probably  find  free  comple- 
ments with  which  to  unite  in  the  serum  of  the  animal  undergoing 
injection. 

While  the  force  of  these  and  of  possibly  other  objections  can  not  be 
denied,  it  would  seem  evident  from  the  experiments  herein  reported 
that  the  only  difference  between  the  behavior  of  immune  serum  on 
the  one  hand  and  of  normal  serum  on  the  other,  upon  dilution,  was  a 
difference  of  degree  and  not  of  kind.  If  this  is  true,  the  theory  of 
complement  diversion  must  be  applicable  to  both  or  to  neither. 

Evidently  there  ate  only  three  possibilities  in  regard  to  the  relative 
number  of  amboceptors  and  of  complements  in  an}'^  giv^en  serum — either 
there  is  just  the  proper  proportion  of  amboceptors  to  complements  or 
there  is  an  excess  of  the  one  or  of  the  other  of  these.  If  the  ambo- 
ceptors and  complements  are  present  in  just  the  proper  proportion, 
then  there  would  be  a  simple  relation  between  the  undiluted  and  the 
diluted  serum.  The  serum  would  become  relatively  and  actualh'  less 
and  less  potent  in  exact  ratio  to  the  extent  of  the  dilution.     If  a  given 


66  BACTKRIOLYTIO    POWER   OF    BLOOD   SERUV   OF    H008. 

nuinlM^r  of  bacteria  were  killed  by  a  certain  amount  of  the  undiluted 
Horuni.  ii  ('(M-tain  fniction  of  this  nuinlK»r  would  l>e  dostroycd  by  the 
same  fraction  of  the  scrum.  If  the  complement  is  in  excess,  evidently 
the  same  rule  would  hold;  the  free  complements  would  neither  help 
nor  hinder  the  proceas.  Hut  if  there  were  an  excess  of  aml>oceptorH, 
a  imrt  of  the  Imcteriii  corres{K)ndin^  to  the  amount  of  this  excess 
would  esca|>e  Imcteriolysis,  accordinjif  to  the  theory  of  complement 
diversion.  On  dilution,  however,  there  would  Im*  relatively  more  bac- 
teria killed  where  there  are  free  amlKM-eptoi-s  present  in  the  undiluted 
serum,  as  already  cxplaine<l  in  the  description  of  the  Neisser-Wechsl)erg 
theory.  gfe 

Since  none  of  the  ex|^'riment«  in  the  present  investi^ition  have  Iwen 
made  to  determine  whether  the  serum  from  normal  hog's  blood  con- 
tains free  amboceptors,  and  no  experiments  of  the  kind  with  hog's 
blood  seem  to  have  been  so  far  reported,  the  matter  nuist  for  the 
present  l)e  left  undecided.  But  the  assumption  of  such  a  condition  in 
the  normal  serum  would  seem  necessary  to  account  for  the  result* 
obtained. 

A  specific  example  taken  from  one  of  the  experiments  already 
described  ma}'  serve  to  show  the  application  of  the  theory  of  complement 
diversion  to  the  phenomena  observed  on  diluting  normal  hog  sera  in  so 
far  as  the  effect  of  dilution  u[K)n  the  bacteriolytic  potency  is  concerned. 
Into  the  undiluted  serum  from  the  blood  of  hog  1S55),  and  into  each 
of  the  various  dilutions,  there  were  introduced  l)efore  injection  of  the 
animal  3,760  bacilli  of  the  Crawford  strain  i)er  1  c.  c.  of  the  serum. 
The  number  of  l>acilli  surviving  aft-er  twent^'-four  hours  in  the  undi- 
luted serum  was  8(>  jK'r  I  c.  c.  There  are  two  conditions  which  may 
be  assumed  to  exist  in  the  serum,  either  of  which  would  account  for 
the  escape  of  the  80  Imcilli,  disregarding  for  the  prasent  the  fact  else- 
where discusse<l  that  there  is  a  multiplication  of  the  bacteria  surviving 
iMicteriolysis  and  assuming  that  the  80  bacteria  in  the  present  case 
represent  mei-ely  thost^  bacteria  which  escaiH»d  bacteriolysis.  Either 
there  were  not  enough  aml)oceptor-complements  for  all  the  Iwcteria 
intro<luced — in  other  words,  there  were  80  short  of  these—  or,  if  there 
were  enough  or  more  than  enough  aml>oceptor-complements,  there 
were  also  80  free  aml>oceptors.  Hither  of  these  two  conditions  would 
acct)unt  for  the  escSajX)  of  the  80  bacteria  in  the  experiment.  A  simple 
calculation  from  the  result  obtained  on  diluting  the  KM-um  will  show 
which  of  these  conditions  is  most  likely  to  have  prevailed.  If  there 
had  l)een  a  paucity  of  amlK>ceptor-complements  -  if  there  had  l)een, 
in  other  words,  just  3,680  of  these  instead  of  over  3,76(>,  which  was 
the  nimd)er  of  bacteria  added-  then  a  dilution  of  1  part  of  the  serum 
to  U  jMirts  of  salt  solution  should  have  killed  only  368  bacilli  |)«'r  1  e.  c. 
when  these  were  introduced;  whereas,  as  a  matter  of  fact,  this  dilu- 
tion killed  2,60«>  |)er  1  c.  c.     There  would  ap|)ear,  therefore,  no  ground 


CONDITIONS    AFFECTING    BACTERIOLYSIS.  57 

for  assuming  the  failure  of  bacteriolysis  in  tlie  immune  serum  to  have 
been  due  to  a  paucity  of  amboceptor-complements  in  the  undiluted 
serum. 

Suppose,  now,  that  the  other  condition  mentioned  above  existed  in 
the  serum,  namely,  that  there  was  an  excess  of  amboceptors  over  and 
above  the  amboceptor-complements,  and  that  there  were  more  ambo- 
ceptor-complements than  the  bacteria  introduced.  A  simple  calcula- 
tion will  show  that  such  a  supposition  will  readily  account  for  the 
results  obtained  in  the  diluted  serum  as  compared  with  the  results  in 
the  undiluted.  If  there  were  in  the  undiluted  serum  26,000  ambocep- 
tor-complements and  80  free  amboceptors  per  1  c.  c.  then  on  dilution  of 
1  to  10  there  would  evidently  be  2,600  amboceptor-complements  and  8 
free  amboceptors  per  1  c.  c.  of  the  diluted  serum.  Consequently  8 
bacteria  would  escape  bacteriolj^sis  on  account  of  being  attached  to  the 
free  amboceptors,  but  2,600  would  be  destroyed. 

The  above  calculations  are,  of  course,  not  intended  to  indicate  pre- 
cisely the  processes  involved,  for  these  are  probabl}'^  complicated  by 
the  multiplication  of  the  surviving  bacteria,  as  mentioned  above. 
Still,  even  if  such  a  complication  is  admitted,  the  probabilities  in  favor 
of  the  condition  last  named  become  even  greater,  as  a  moment's  con- 
sideration will  show. 

A  further  complication  may  also  arise  from  the  possibility,  however 
.remote,  that  the  amboceptors  and  complements  are  not  uniforml}'  dis- 
tributed in  the  serum — that  while  they  may  perhaps  usually  be  so 
distributed,  they  may  be  found  more  abundantly  in  one  part  of  the 
serum  than  in  another. 

SUMMARY   OF   THE   CONDITIONS   AFFECTING   BACTERIOLYSIS. 

The  conditions  under  which  bacteriolysis  would  take  place,  and 
those  under  which  no  bacteriolysis  would  take  place  in  any  given 
serum,  may  be  summarized  as  follows: 

1.  Complete  bacteriolysis  could  take  place  only  where  there  were 
no  free  amboceptors  and  where  there  were  at  the  same  time  a  number 
of  amboceptor-complements  equal  to  or  greater  than  the  number  of 
bacteria  introduced. 

On  dilution  in  a  serum  of  this  kind  there  would  be  a  loss  of  bac- 
teriolytic power  in  proportion  to  the  degree  of  dilution  if  the  ambo- 
ceptor-complements were  exactly  equal  in  number  to  the  bacteria 
introduced.  If  there  were  more  amboceptor-complements  originalh' 
in  the  undiluted  serum  than  the  bacteria  introduced,  then  on  dilution 
there  would  be  relatively  more  bacteria  destroyed.  If  the  excess  of 
amboceptor-complements  is  large  enough,  there  could  of  course  be 
enough  present  in  the  diluted  serum  to  kill  as  many  bacteria  as  were 
killed  by  the  serum  before  dilution. 


58     BACTERIOLYTIC  POWKR  OK  BLOOD  HKRUM  OF  H008. 

2.  PHrtiiil  Imrtoriolysih  wuuld  follow  when  there  wero  fewer  hiiiIm)- 
oeptors  prt>HCiit  in  the  serum  timn  the  I>act4>ria  intriKliiciHl  ami  when 
ftt  the  sjiujo  time  there  wore  any  umlxK'eptorM-omplements  i)re.sent. 

The  extent  of  lmet«riolysis  ujxjm  dilution  would  depend  ujx>n  the 
nuinher  of  amlKK^eptor-complements  pres<»nt  originally. 

H.  No  l»acteriolysis  could  tjike  plaee  if  the  free  amlKxeptors  \\»n' 
e<jUHl  in  nuiulNM*  to  the  Imcteria  introduced,  or  if  they  were  in  exce>.> 
of  this  numlH>r,  either  in  the  undiluted  or  the  diluted  nenuiL 

THE  THI-X^RY   OF  COMPLEMENT   l)IVEK«ION. 

In  spite  of  the  serious  difficulties  alluded  to  it  would  nevertheless 
app<»ar  that  the  theory  of  complenient  diversion  {^ives  an  insight  into 
the  process  of  bacteriolysis  occuring  in  normal  serum  in  so  far  as  this 
is  atfected  l>v  dilution — in  those  cases  at  least  where  the  ex[)eriments 
show  that  the  serum  is  a<tually  or  relatively  more  potent  upon  dilution 
than  when  undiluted-  irrespective  of  the  nature  of  the  serum,  whether 
this  is  normal  or  inunune.  But  this  would  leave  the  results  obtained 
in  certain  other  experiments  still  unexplained.  It  will  Ik?  recalled 
that  in  ciM'tain  experiments  the  serum  was  found  to  be  Imctericidal 
when  undiluted,  that  it  lost  in  bactericidal  potency  on  dilution,  but 
that  it  gained  in  potency  upon  further  dilution.  It  is  tho«e  results 
which  would  l)e  left  unexplained  by  the  theory  of  conj])lement  diver- 
sion as  thus  far  discussed.  One  example  of  this  behavior  among  others 
is  to  \h}  seen  in  Table  XXVI,  in  the  serum  of  hog  17J>8,  ten  days 
after  the  inoculation  of  the  animal  with  the  F.  2i)  strain  of  Ji.  cliolerie 
8ui«.  In  one  of  the  tests  there  were  introduced  into  the  undiluted  serum 
and  into  the  vaiious  dilutions  in  round  numbers  8(M)  Crawford  Iwicilli. 
The  result  after  twenty-four  hours  was  that  all  the  bacilli  were 
destroyed  in  the  undiluted  serum,  while  in  the  dilutions  of  1  to  50  and 
1  to  50)  there  was  an  increase  of  about  thirty-three  fold  and  one 
hundredfold,  resi^ectively.  Rut  in  the  dilution  of  1  to  5,00o  there 
were  onl}'  140  liacilli  remaining.  In  other  words,  the  undiluted 
.serum  showed  strongly  Imctericidal  power,  and  the  dilution  of 
1  to  5i<MK)  also  showed  considerable  potency,  while  the  dilutions 
l>etween  these  two  showed  at  most  but  feeble  power.  If  this  result  is 
analyzed,  it  will  Ihj  seen  that  the  serum  ap|3<Mired  to  act  like  normal 
serum  at  the  start  when  undiluted  and  in  the  first  dilutions,  but  that 
on  further  dilution  it  apjM'ared  to  act  like  immune  serum.  This  sug- 
gests that  possibl}'  the  normal  aml)i>ceptor-complements  persisting  in 
the  serum  may  have  l»een  more  |K)teiit  than  the  inunune  amlK>ceptors 
formed  in  conse(juence  of  the  injection,  that  the  tirst  t'trert  of  dilution 
was  to  reduce  the  |)otency  of  the  nonnal  amlKK-eptor-i-omplements,  and 
that  further  dilution  enabled  the  imnunie  aml>oceptors  to  pr<Kluce  the 
phenomenon  of  complement  diversion.  This  would  l>e  <|uite  in  accord 
with  certain  views  expressed  elsewhere  in  this  pa|)er  to  the  effect  that 


THEORY    OF    COMPLEMENT   DIVERSION.  59 

the  injections  of  cultures  or  their  products  while  they  cause  the  for- 
mation of  new  amboceptors  leave  the  normal  amboceptor- complements 
unaffected. 

In  the  above  discussion  it  is  assumed  that  the  two  bodies  concerned — 
the  amboceptor  and  the  complement  of  Ehrlich,  the  sensibilisatrice 
and  the  alexin  of  Bordet — are  capable  of  uniting  and  do  actually 
unite  independently  of  the  presence  of  bacteria  or  of  other  cells.  But 
Bordet'  has  recently  published  a  series  of  investigations  tending  to 
show  that  the  experiments  of  Ehrlich  and  Sachs'',  which  constitute  the 
chief  evidence  in  favor  of  this  view,  are  capable  of  quite  a  different 
interpretation  from  this,  and  that  this  interpretation  is  in  fact  not  justi- 
fiable from  the  results  of  the  experiments  which  consisted  in  the 
demonstration  of  the  fact,  not  denied  by  Bordet,  that  ox  serum  will 
produce  cytolysis  only  when  the  serum  has  in  it  amboceptors  and  com- 
plements simultaneously.  It  is  not  possible,  as  in  some  other  cases,  to 
produce  cytolysis  by  sensitizing  cells  with  ox  amboceptors — that  is, 
with  heated  ox  serum — and,  after  washing  these  sensitized  cells,  add- 
ing complement — that  is,  fresh  serum.  Cytolysis  with  ox  serum 
takes  place  only  when  the  heated  ox  serum  and  some  unheated  fresh 
serum  (horse  serum  was  the  kind  used  in  the  experiments)  are 
employed  at  the  same  time.  This  is  interpreted  by  Ehrlich  and  Sachs 
as  showing  that  while  the  free  amboceptors  present  in  the  ox  serum 
will  not  unite  with  the  cells  they  will  and  do  so  unite  when  they  are 
previously  attached  to  complements.  But  Bordet's  results  appear  to 
show  quite  plainly  that  in  this  case  the  horse  serum  which  was  used 
as  complement  produces  cytolysis  quite  independently  of  the  ox  serum, 
and  that  while  cytolysis  takes  place  more  promptly  when  heated  ox 
serum — ox  amboceptors — are  added,  the  ox  serum  is  not  necessar}'. 
Bordet  therefore  regards  the  experiments  as  showing- that  the  heated 
ox  serum  acted  merel}'^  as  an  accelerator  of  cytolysis. 

Bordet  summarizes  his  conclusions  as  follows: 

We  see  but  one  rational  explanation  of  the  peculiar  action  of  ox  serum — that  there 
exists  in  the  serum  a  peculiar  substance  capable  of  resisting  heat  of  56°  C.  and 
which  remains  unaltered  for  many  months  in  this  heated  serum.  The  substance 
is  probably  of  an  albuminous  or  colloid  character,  and  does  not  adhere  to  the 
normal  corpuscles,  but  is  precipitated  upon  the  corpuscles  which  are  previously 
charged  with  sensibilisatrice  and  alexin.  We  believe  that  it  is  a  veritable  process 
of  glueing  of  absorption  depending  upon  molecular  adhesion.  *  *  *  In  con- 
formity with  the  statements  of  Ehrlich  and  Sachs,  experiments  show  that  the 
corpuscles  of  guinea  pigs  become  hemolyzed  in  a  mixture  of  fresh  horse  serum  and 
of  ox  serum,  the  latter  having  been  heated  to  56°  C,  while  they  resist  hemoly- 
sis if  they  are  first  subjected  to  the  action  of  the  heated  ox  serum  and  have  the  horse 
serum  added  subsequently.  But  the  interpretation  offered  by  Ehrlich  and  Sachs, 
according  to  which  the  sensibilisatrice  furnished  by  the  ox  serum  does  not  unite 
with  the  corpuscles  unless  it  (the  sensibilisatrice)  is  previously  connected  with  alexin 
derived  from  the  horse  serum  is  nek  correct.  In  the  first  place,  the  sensibilisatrice, 
which  plays  a  preponderating  and  most  essential  role,  is  not  contained  in  the  ox 


60  BACTKRIOLYTIO   POWER   OK    BL<K>D   AKRUM    OK    HOQft. 

iwnini  at  all,  but  is  fnrniMhed  by  the  home  aerutu.  ConMMiiivntly  these  HeiiHibili- 
satriit*  )tehuv«>  like  all  of  tht'ir  i-ongt'iuTH,  in  tin-  nemH!  that  tlicy  «lo  not  rtHjuin*  the 
jirttH'mi*  of  the  alexin  Ix^fore  they  are  caitahle  of  uniting  with  the  ror|»u»»clee. 

Finally,  this  interpretation  li'aves  r«>ni|>letely  in  the  dark  the  very  Hftecial  fjeculiari- 
tit*x  of  the  au*'»  of  heniolvHis  in  question. 

The  i»eeuliarity  t)f  ox  wTurn  conHist^  in  the  presenile  of  a  «t»rtain  element  which 
n>t<iMt8  hd"  C,  an«l  al^>o  rt«ixtM  Mtanding,  and  i»  of  the  natun*  of  a  <!olloid,  doubtle(W 
albuminoid,  and  whieh,  fiirtherraort*,  in  ubHorlted  by  cori)U«'le«  which  have  liecome 
chargtMl  with  HenttibiliHatrice  and  alexin,  but  which  remainn  free  in  the  preeeni-e  of 
nonnal  corpnwlos  or  of  corpuscles  nu'reiy  HcuHitiztMl — i.  p.,  (*orpu8clet4  treate<l  with 
heat«-<l  Hi>mm  alone.  The  alwHjrption  of  this  colloid  by  corpuscles  which  have  lieen 
tn-atcil  with  l)oth  m>nHibili8atrice  un«l  alexin  hiw  the  effect  of  energetically  aggluti- 
nating  them  antl  of  rendering  them  more  susceptible  to  hemolysis  except  ander  cer- 
tain cin-umBtiinces,  •  »  *  The  absorptiiin  of  the  Bensitized  and  alexinize<l  cor- 
puscles is  very  lik«'ly  due  tt)  molecular  adhesion,  the  preliminary  treatment  having 
nuMlitied  the  <-orpu8cl«*8  in  ho  far  an  their  aflhesive  properties  are  coni"eme<l.  I'nder 
these  conditions  the  absorption  may  take  place  indefiendently  of  the  species  of 
animal  from  whi«-h  the  corpuK-les  are  obtained;  it  may  even  take  place  with  the 
corpU8<'lefl  of  the  same  animal  which  furnieheH  the  colloiil.as  in  the  case  of  ox  senim. 

Now,  if  the  contention  of  IJordet  as  set  forth  above  is  correct,  and 
if  the  two  bodies  concerned  in  cytolysis  do  not  unite,  then  of  course 
the  theory  of  complement  diversion  must  fall,  since  it  requires  tis  the 
first  condition  the  union  of  ambocteptors  with  complements.  Never- 
theless, there  appears  to  be  as  yet  no  other  explanation  of  the  phe- 
nomena observed  on  diluting  immune  serum,  and,  as  an  attempt  ha** 
been  made  to  show  in  the  present  paper,  there  seems  to  be  no  other 
explanation  of  the  behavior  of  normal  serum  on  dilution. 

CONCLUSIONS. 

In  summarizing  the  results  of  the  present  investigations  it  is  seen — 

1.  That  the  bactericidal  potency  of  the  serum  from  the  same  hog 
varies  from  time  to  time. 

2.  That  the  serum  from  one  and  the  same  drawing  dilTers  in  potency 
for  different  strains  of  B.  choleras  ttuiif. 

3.  That  while  the  effect  of  standing  is  to  weaken  the  potency,  this 
effect  is  more  marked  in  some  specimens  of  serum  than  in  others 
under  the  sjune  conditions. 

4.  That  the  bactericidal  power  of  the  serum  from  the  venous  blood 
of  hogs  is  not  always  more  potent  than  that  from  the  arterial  blood. 

5.  That  heating  the  serum  at  alK>ut  54"  C.  for  thirty  minutes  inacti- 
vates the  serum. 

6.  That  the  Nei.sser-Wechsberg  phenomenon  is  .sometimes  seen  in 
and  sonietimes  missed  from  the  blood  of  hogs  injected  with  cultures  of 
B.  cholrrse  Hui». 

7.  That  the  theory  of  complement  diversion  may  not  only  account 
for  the  behavior  of  mmume  serum,  but  also  for  that  of  normal  serum 
on  dilution,  or,  at  least,  that  the  theory,  if  applicable  in  the  one  case, 
is  equally  so  in  the  other. 


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61 


68  RIHLKM}RAI>HY. 

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l't'l»orantiY)akteri(>lytiM(>he(antaKoniHti8chc)  Hutwtanxen  norinalvr  wra.  Dent, 
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(17)  IhrairFKR,  R[irH.\Ri>],  and  Wakhbrman.v,  A[i'orHT]. 

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(20)  Wolff,  Alfred. 

■   Uel)er  gmndgesetze  der  immunitat.    Cent  f.  bakt,  abt  1,  Orig.,  bd.  37,  na 
4,  p.  666-576.     Jena,  Dec.  12,  1904. 

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